Methods of use of fibroblast growth factor, vascular endothelial growth factor and related proteins in the treatment of acute and chronic heart disease

ABSTRACT

Disclosed herein is a rational, multi-tier approach to the administration of growth factor proteins in the treatment of heart disease. Also disclosed is a method to evaluate the effectiveness of the administration of growth factor proteins comprising the clinical assay of CPK-MB levels in a patient undergoing treatment with growth factor proteins. In addition, there is disclosed a method for treatment of heart disease comprising administration of a therapeutically effective amount of a growth factor protein by oral inhalation therapy.

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under Title 35, U.S.C. § 119(e)of United States Application No. 60/195,624, Filed Apr. 6, 2000.

FIELD OF THE INVENTION

[0002] The present invention relates generally to strategies and methodsfor the treatment of chronic and acute heart disease through thedelivery of one or more related protein growth factors such asfibroblast growth factor (FGF) and vascular endothelial growth factor(VEGF).

BACKGROUND OF THE INVENTION

[0003] Chronic myocardial ischemia is the leading cardiac illnessaffecting the general population in the Western world. Since theoccurrence of angina symptoms or objective physiological manifestationsof myocardial ischemia signifies a mismatch between myocardial oxygendemand and the available coronary blood flow, the goal of therapy is torestore this balance. This can be achieved either by attempting toprevent further disease progression through modification of riskfactors, or by more aggressive modes of therapy such as reducing themyocardial oxygen demand (i.e. reducing the heart rate, myocardialcontractility or blood pressure) by using anti-anginal medications, orby restoring the blood supply by means of mechanical interventions suchas percutaneous transluminal angioplasty or its variants, or coronaryartery bypass surgery, coronary angioplasty (PTCA) or bypass surgery(CABG). When CABG is selected as the treatment option, its success maybe limited by the inability to provide complete revascularization inthose patients in whom the artery that supplies a viable butunderperfused myocardial territory is not graftable because ofdiffuse-disease, calcifications, or small size. Completerevascularization cannot be achieved in up to 37% of patients undergoingCABG. This number is probably much lower today. However, patients whoundergo complete revascularization have improved 5-year survival andangina-free survival compared with patients who have incompleterevascularization. Therefore, an adjunctive treatment strategy iswarranted in patients undergoing CABG if complete revascularization isnot possible. Percutaneous catheter-based revascularization is oftenprecluded secondary to the same attributes that made the myocardialterritory ungraftable: diffuse disease and small or calcified vessels.

[0004] The field of angiogenesis research was initiated 30 years ago bya hypothesis that tumors are angiogenesis-dependent. Folkman, J. “Tumorangiogenesis: therapeutic implications.” N. Engl. J. Med. 285: 1182-1186(1971). Shortly thereafter, in the early 1970's, it became possible topassage vascular endothelial cells in vitro for the first time.Bioassays for angiogenesis were developed subsequently through thatdecade. The early 1980's saw the purification of the first angiogenicfactors. Clinical applications of angiogenesis research are beingpursued along three general lines: 1) prognostic markers in cancerpatients; 2) anti-angiogenic therapy (in cancer treatment); and 3)angiogenic therapy (treatment of heart disease).

[0005] In discussing the field of angiogenesis, it is important todifferentiate 3 different processes that contribute to the growth of newvessels. Vasculogenesis is the primary process responsible for thegrowth of new vasculature during embryonic development, and it may playan as yet undefined role in mature adult tissues. Arteriogenesis refersto the appearance of new arteries possessing fully developed tunicamedia, while true angiogenesis describes the growth of collateral-likevessels lacking the development of media. In the case of coronarycirculation, arteriogenesis is usually taken to mean new,angiographically visible epicardial vessels while angiogenesis refers tothin-walled intramyocardial collaterals.

[0006] Occlusion of coronary arteries is often associated withdevelopment of collateral circulation in patients with atherosclerosis.Although the existence of collateral circulation in such patients isassociated with improved clinical outcomes, the net effect is rarelyadequate to compensate fully for the flow lost to occlusion of nativeepicardial coronary arteries. A number of growth factors have beenassociated with myocardial and peripheral limb ischemia, particularlybasic fibroblast growth factor (bFGF), acidic fibroblast growth factor(FGF-1), and vascular endothelial growth factor (VEGF), which have beenshown to induce functionally significant angiogenesis in animal modelsof myocardial and limb ischemia. These promising preclinical resultshave rapidly lead to the study of these growth factors in patients withchronic myocardial ischemia using intracoronary (IC), intravenous(i.v.), and local delivery (myocardial injection).

[0007] Therapeutic myocardial angiogenesis is a novel approach to thetreatment of myocardial ischemia based on the use of proangiogenicgrowth factors to induce the growth and development of new blood vesselsto supply the myocardium at risk. Angiogenesis is a complex processinvolving endothelial and smooth muscle cell proliferation andmigration, formation of new capillaries, and extracellular matrixturnover. Various heparin-binding growth factors, including basicfibroblast growth factor (FGF-2), acidic fibroblast growth factor, andvascular endothelial growth factor (VEGF) induce angiogenesis in chronicmyocardial ischemia. Given the typically long time course of newcollateral vessel development, most attempts to stimulate myocardialangiogenesis have used methods of prolonged growth factor delivery,including gene therapy, continuous infusions, repeated injections, orsustained release polymers. However, some of these options are notfeasible or practical in patients with ischemic heart disease, makingsingle dose administration, if effective, a potentially superiorstrategy in these patients.

[0008] Angiogenesis is a complex process that involves endothelial cellmigration and proliferation, extracellular matrix breakdown, attractionof pericytes and macrophages, smooth muscle cell proliferation andmigration, formation and “sealing” of new vascular structures, anddeposition of new matrix. A number of growth factors, including thefibroblast growth factors (FGF) and vascular endothelial growth factors(VEGF) are integrally involved in the angiogenic response in ischemicconditions and in certain pathological states. The availability of thesefactors has led to studies, which have demonstrated a therapeuticbenefit in various animal models of acute and chronic myocardialischemia. In particular, basic fibroblast growth factor is an attractivecandidate as an agent for therapeutic angiogenesis.

[0009] The therapeutic goal of attempting to ameliorate chronic ischemicconditions through revascularization by administration of variousprotein growth factors is feasible only due to the chronic nature of thecondition and the resulting long-term time scale for treatment. In acuteclinical situations, such as myocardial infarct, or therapeuticprocedures likely to lead to reperfusion injury, the luxury of long timescales for revascularation is not available. However, theadministration, via various routes, of growth factors such as FGF hasbeen demonstrated to be effective in reducing the effects of myocardialinfarct within a time frame that precludes a therapeutic contributionfrom the angiogenic function of these proteins. See, for example, myearlier U.S. Pat. No. 4,296,100, the disclosure of which is herebyincorporated specifically by reference. Thus, by a mechanism yet to beelucidated, protein growth factors such as FGF and VEGF and relatedproteins are capable of demonstrating a therapeutic utility insituations involving acute damage to the heart.

SUMMARY OF THE INVENTION

[0010] In a first embodiment, the present invention provides a methodfor the systematic, multi-tiered treatment of heart disease by deliveryof therapeutic growth factor proteins comprising the steps of a.)selecting a patient displaying symptoms of heart disease; b.)administering at least one dose of an effective amount of a firsttherapeutic growth factor protein formulation by oral inhalation; c.)monitoring levels of CPK-MB in the patient; d.) determining whetheradministration of the growth factor protein formulation was effective intreating the symptoms of heart disease in the patient; e.) administeringone or more additional doses of a second growth factor proteinformulation by a method of delivery more invasive than delivery by oralinhalation; and f.) repeating steps c.) through e.) until there is aclinical indication of amelioration of the symptoms of heart disease inthe patient, or until there is a contraindication to continuedtreatment. Preferably, the protein formulation comprises a growth factorprotein selected from the group consisting of FGF-1, FGF-2, VEGF, andmixtures thereof. In one aspect of this embodiment, the method of theinvention contemplates application where the symptoms of heart diseaseare acute. These acute symptoms of heart disease can be brought on by acondition selected from the group consisting of myocardial infarct,unstable angina, an acute anginal attack, and reperfusion injury.Furthermore, the reperfusion injury is induced by a procedure selectedfrom the group consisting of thrombolytic therapy, bypass surgery andangioplasty. Alternatively, the method of the present inventioncontemplates applicatin where the symptoms of heart disease are chronic.

[0011] In an alternative embodiment, the method of the presentencompasses the administration of therapeutic amounts of a growth factorprotein formulation in the treatment of heart disease by delivering theprotein formulation by inhalation therapy. Preferably, in the practiceof the present invention, the protein formulation is a dry powderformulation. Alternatively, the protein formulation is a liquid aerosolformulation.

[0012] In another embodiment, the present invention provides a methodfor monitoring clinical effectiveness of administration of a growthfactor protein formulation in the treatment of heart disease, the methodcomprising the steps of obtaining a sample of a biological fluid from apatient displaying symptoms of heart disease; performing an assay of thebiological fluid to determine an amount of CPK-MB present in the fluid;administering a therapeutic amount of a growth factor proteinformulation to the patient; and repeating the last two steps until theassayed amount of CPK-MB in the biological fluid has decreased by anamount sufficient to indicate the clinical effectiveness of theadministration of the growth factor protein formulation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is an illustration of the lung, indicating a mechanism ofdelivery of aerosol drug particles through the lung and into thebloodstream.

[0014]FIG. 2 is an illustration of the results of measured regional wallthickening in the LAD (normal) and LCX (collateral-dependent)distribution.

[0015]FIG. 3 is an illustration of, at top, MRI perfusion images of theleft ventricle and, at bottom, the ischemic zone extent in all groups oftest animals.

[0016]FIG. 4 is an illustration of histopathological sections from theLCX distribution demonstrating an increased number of capillaries in alltreatment groups.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Recent advances in growth factor therapy for the treatment ofischemic disease of the heart and peripheral vasculature offer hope of anovel treatment strategy that is based on generation of new blood supplyin the diseased heart. Members of the fibroblast growth factor family,vascular endothelial growth factor family, and several other moleculeshave all been shown to result in functionally significant angiogenesisin animal models of acute and chronic myocardial and peripheral limbischemia. The promising preclinical data have propelled the use of theseangiogenic growth factors in clinical studies of ischemic heart andperipheral vascular disease. These growth factors are presumed to induceneovascularization by stimulating endothelial and smooth muscle cellproliferation and migration, dissolving the extracellular matrix,attracting pericytes and macrophages, and finally forming and “sealing”new vascular structures with deposition of new matrix.

[0018] In the surgical treatment of acute ischemic conditions,approximately 500 000 PTCA and 375 000 CABG procedures are performedannually in the United States. However, a significant number of patientsare suboptimal candidates for CABG or PTCA or do not achieve completerevascularization with these procedures. These patients would likelybenefit from additional measures to achieve enhanced revascularization,and therapeutic angiogenesis may serve this role. Several studies havedemonstrated that chronic administration of FGF-2 results in significantmyocardial angiogenesis in animal models of myocardial ischemia andinfarction. However, because of the protracted time course required fornew collateral vessel development, many attempts to stimulate myocardialangiogenesis have used methods of prolonged growth factor delivery,including gene therapy, continuous infusions, repeated injections, andsustained release polymers. Many of these therapeutic strategies,particularly those requiring repeated access or major surgicalintervention, are impractical, or less than ideal, from a clinicalstandpoint. The pericardial space offers potentially unique advantagesin convenience, safety, and efficacy as a cardiovascular drug depot sitefor the administration of proangiogenic growth factors.

[0019] In investigating the effects of a single intrapericardialinjection of increasing FGF-2 doses in a porcine model of chronicmyocardial ischemia, separate saline and heparin control arms were usedto address the potential angiogenic effects of heparin alone or incombination with FGF-2. However, no significant differences were foundbetween the heparin (at the dose used) and saline arms in any of themeasured parameters. Intrapericardial FGF-2, on the other hand, resultedin an improvement in left-to-left angiographic collaterals, occluded LCXcoronary artery blood flow, LCX (ischemic territory) myocardialperfusion, and LCX regional wall function as measured by MRI.Improvements in ischemic territory regional wall function and myocardialperfusion were positively correlated with FGF-2 dose, with nearnormalization of wall function and perfusion in the 2 mg FGF-2 group.Qualitative histopathologic examination showed increased myocardialvascularity in FGF-2-treated animals without any adverse findings.

[0020] In considering growth factor-induced neovascularization, it isimportant to distinguish intramyocardial collateral development fromformation of epicardial collaterals (neoarteriogenesis). The process ofintramyocardial collateral-development (angiogenesis) is characterizedby appearance of thin-walled vessels with poorly developed tunica mediagenerally under 200 μm in diameter and by an increase in the number oftrue capillaries (<20 μm in diameter containing only a singleendothelial layer), whereas the neoarteriogenesis is characterized bydevelopment of larger vessels (>200 μm in diameter) with well developedtunica media and adventitia that usually form close to the site of theocclusion of a major epicardial coronary artery (bridging collaterals)or extend from one coronary artery to the other. The distinction betweenthese two groups of newly formed vessels is important not only from thepoint of view of their location but also because stimuli for theirdevelopment appear to be quite different and because they may exhibitdifferent physiological properties. It is unclear whetherintrapericardially administered FGF-2 exerts its beneficial effects onmyocardial revascularization by acting on the epicardial surface (whereit is in greatest concentration) to induce collateralization aroundsites of occlusion in the epicardially situated major coronary arteries,or whether it diffuses into the myocardium and myocardialmicrocirculation to induce angiogenesis at a more microscopic level, orboth. However, the demonstrated effectiveness of the low-dose (30 μg)intrapericardial FGF-2 suggests that the presence of FGF-2 on theepicardial surface may play a key role in inducing functionallysignificant angiogenesis.

Fibroblast Growth Factors

[0021] Acidic fibroblast growth factor (aFGF), also referred to asFGF-1, is a monomeric, acidic protein of approximately 18 kDa. It sharesabout 55% homology with the basic protein FGF-2. Both are prototypes forthe FGF family members and their three dimensional structure are known.

[0022] Basic fibroblast growth factor (bFGF), also referred to as FGF-2,is a 16.5Kd 146 amino acid protein that belongs to the FGF family, whichnow comprises more than 22 structurally related polypeptides. One of thekey differences between the various FGFs is the presence or absence ofthe leader sequence required for conventional peptide secretion (absentin FGF-1 and FGF-2). Another difference is the varied affinity for thedifferent isoforms of FGF receptors. As for most heparin-binding growthfactors, bFGF binds with high affinity to cellular heparin sulfates and,with even higher affinity, to its own tyrosine kinase receptors (FGFreceptors 1 and 2). The ability of bFGF to bind cell surface and matrixheparin sulfates serves both to prolong its effective tissue half-lifeand to facilitate its binding to the high affinity receptors. While bFGFis present in the normal myocardium, its expression is stimulated byhypoxia and hemodynamic stress.

[0023] FGB-2 is a pluripotent mitogen capable of stimulating migrationand proliferation of a variety of cell types including fibroblasts,macrophages, smooth muscle and endothelial cells. In addition to thesemitogenic properties, FGF-2 can stimulate endothelial production ofvarious proteases, including plasminogen activator and matrixmetalloproteinases, induce significant vasodilation through stimulationof nitric oxide release and promote chemotaxis. FGF-2 is present in thenormal myocardium and its expression is potentiated by hypoxia orhemodynamic stress. Because of its heparin-binding properties, FGF-2binds avidly (Kd 10⁻⁹M) to endothelial cell surface heparin sulfates.This interaction serves to prolong effective tissue half-life of theFGF-2 protein, facilitates its binding to its high-affinity receptorsand plays a key role in stimulation of cell proliferation and migration.BFGF also possesses a plethora of other biological effects such as theability to stimulate NO release, to synthesize various proteases,including plasminogen activator and matrix metalloproteinases, and toinduce chemotaxis. Homozygous deletion of the bFGF gene is associatedwith decreased vascular smooth muscle contractility, low blood pressureand thrombocytosis. One interesting aspect of bFGF is its biologicalsynergy with VEGF. Thus, a combination of BFGF and VEGF is far morepotent than bFGF alone in inducing angiogenesis in vitro and in vivo.Furthermore, bFGF induces VEGF expression in smooth muscle andendothelial cells.

[0024] Despite significant levels of bFGF in normal tissues, the growthfactor does not appear to be biologically active, as suggested by thelack of on-going angiogenesis. While the precise explanation for thislack of activity of the endogenous bFGF is uncertain, contributingfactors probably include very low levels of expression of FGF receptors1 and 2 and syndecan-4, another transmembrane protein involved inbFGF-dependent signaling. In addition, endogenous bFGF may besequestered in the extracellular matrix by binding to heparinsulfate-carrying proteoglycan percelan and, thus, be unavailable to bindto its signaling receptors.

Vascular Endothelial Growth Factor

[0025] Similar to bFGF, vascular endothelial growth factor (VEGF)transcripts are detected in all cardiac tissues. VEGF and the expressionof its receptors in the heart are induced 7-fold by hypoxia/ischemia.The unique feature of VEGF was thought to be the narrow spectrum ofactivity, presumed to be confined to endothelial cells because of therestricted expression of its receptors. However, recent studies suggestVEGF receptor expression is more widespread and includes monocytes andsome smooth muscle cells. Furthermore, VEGF is capable of inducing bFGFexpression, thereby further increasing its biological spectrum ofactivity. VEGF is a potent and specific mitogen for vascular endothelialcells that is capable of stimulating angiogenesis during embryonicdevelopment and tumor formation. The VEGF family of structurally relatedgrowth factors has five mammalian members, VEGF, VEGF-B, VEGF-C, VEGF-D,and placenta growth factor (PIGF), all encoded by separate genes.Stacker, S. A. and Achen, M. G. “The vascular endothelial growth factor(VEGF) family: signaling for vascular development.” Growth Factors 17:1-11 (1999).

[0026] Fibroblast Growth Factor (FGF), in its human and bovine basicforms, and in its human acidic form, has been used successfully to treatischemic heart disease, including chronic angina, by stimulatingangiogenesis (the growth of new blood vessels). Vascular EndothelialGrowth Factor (VEGF) has also been used to treat ischemic heart diseaseby stimulating angiogenesis. FGF has been demonstrated to show areduction in acute myocardial infarct size after treatment. Treatment ofmyocardial infarct (MI) requires acute intervention by health careprofessionals, whereas angiogenesis may take at least several days tooccur to a sufficient extent to demonstrate any clinical benefit in theaffected patient. It is the purpose of this invention to utilize a formof FGF and/or VEGF, or other related growth factor proteins, to bringimmediate relief from MI, unstable angina, or an anginal attack andthen, utilizing the same or an alternate delivery system, to promoteangiogenesis for the relief of subacute or more chronic symptoms.

[0027] Angiogenesis begins when blood-starved areas of the heart sendout receptor signals. The purpose of this invention is achieved byadministration to the affected patient of an effective amount of a formof FGF and/or VEGF via inhalation delivery techniques. Inhalationtreatment with FGF and/or VEGF for the management of coronary arterydisease should be successful because the lung is one of the least bloodor oxygen starved organs. The FGF or VEGF would end up on the leftatrium of the heart and from there travel to the coronary arteries whereit would be most useful. The inhalation of FGF and/or VEGF into thelungs could be used for the treatment of MI, unstable angina, or ananginal attack. This delivery system could also be used before, during,and/or after thrombolytic therapy (such as administration of recombinanttissue plasminogen activator) to help alleviate ischemic or reperfusioninjury.

[0028] After successful treatment of acute myocardial infarct or acuteischemia via the methods of the present invention, angiogenesis may alsooccur. If it does not occur within two or three weeks, then theinhalation therapy could be repeated or the FGF and/or VEGF could begiven through a catheter into the coronary arteries or by directinjection in the left atrium, or ventricular myocardium via a limitedthoracotomy. For the treatment of acute myocardial infarct (with orwithout thrombolytic therapy), unstable angina or an anginal attack, theleast invasive method would be preferred. Besides inhalation into thelungs, other available methods of delivery could be sublingual,intranasal, or IV utilizing one of the forms of FGF and/or VEGF. If theleast invasive approaches are not successful in the treatment of acutemyocardial infarct or acute ischemic, then alternate deliver systemsshould be explored. As clinically indicated, the FGF and/or VEGF couldbe given through a catheter into the coronary arteries or by directinjection into the left atrium or ventricular myocardium via a limitedthoracotomy. To assess the efficacy of VEGF or FGF in the treatment ofacute myocardial infarct or unstable angina, you could follow levels ofcreatine phosphokinase-myocardial band (CPK-MB) isoenzymes. Even aminimal elevation above normal range would be considered significant.With treatment, the rise in the level should be less when compared toplacebo.

[0029] Alternative methods of delivery for treatment of coronary arterydisease should also be considered. FGF and/or VEGF could be administereddirectly into the myocardium during transmyocardial laserrevascularization, into the coronary arteries during angioplasty, or byinjection into the coronary arteries, myocardium or bypass grafts duringcoronary bypass surgery. When injected into the myocardium, slow releaseforms of FGF or VFGF should be considered. It might also be possible toinject FGF and/or VFGF into the myocardium via a catheter passed duringcardiac catheterization.

[0030] To promote angiogenesis for the relief of chronic angina orischemia the least invasive method would be preferred. The inhalation ofFGF and/or VEGF into the lungs could be used to achieve this goal.Besides inhalation into the lungs, other available methods of deliverycould be sublingual, intranasal, or IV utilizing one of the forms of FGFand/or VEGF. If the least invasive approaches are not successful inpromoting angiogenesis, then alternate delivery systems should beexplored. As clinically indicated the FGF and/or VEGF could be giventhrough a catheter into the coronary arteries or by direct injectioninto the left atrium or ventricular myocardium via a limitedthoracotomy.

Methods of Delivery of Growth Factors

[0031] Despite promising preclinical data, the progression of angiogenicgrowth factor therapy to the clinical trials stage awaits a practicaldelivery strategy. This requirement essentially eliminates all forms ofprolonged or frequent repetitive intracoronary infusions. Localperivascular delivery is more easily adaptable to clinical trials, butit requires open-chest surgery. One such form of delivery is heparinalginate capsules that provides prolonged (4 to 5 weeks) first-orderkinetics release of the growth factor from the polymer. The capsules areeasily implanted and do not provoke an inflammatory response. Onepotential advantage of perivascular delivery is the absence of theendothelial barrier and the absence of the rapid washout that is typicalwith intravascular administration.

[0032] The pericardial space may potentially serve as a drug deliveryreservoir that might be used to deliver therapeutic agents to the heart.Chronic intrapericardial FGF-2 delivery in a rabbit model of angiotensinII-induced cardiac hypertrophy resulted in a localized myocardialangiogenic response. A single intrapericardial injection of FGF-2 withor without heparin resulted in localized angiogenesis and myocardialsalvage in a canine model of myocardial infarction. Moreover, theconcentration of FGF-2 and VEGF in the pericardial fluid of patientswith unstable angina has been documented to be higher than that inpatients with nonischemic heart disease, suggesting that increases inthe levels of proangiogenic growth factors in the pericardial space mayreflect an endogenous and, indeed, physiological response to myocardialischemia and injury. Accordingly, the pericardium may serve as a usefulreservoir for proangiogenic growth factor administration in patientswith coronary disease.

[0033] An alternative approach to perivascular administration of bFGFinvolves intrapericardial delivery of the growth factor. A majoradvantage of this approach is that it can be accomplished via acatheter, obviating the need for open-chest surgery. However, theclinical application of intrapericardial delivery is limited to a smallnumber of patients currently being enrolled in coronary angiogenesistrials because of the high prevalence (80 to 90%) of prior coronaryartery bypass surgery in this group of patients.

[0034] The feasibility of short duration intracoronary or intravenousinfusions and endomyocardial injections has also been tested in animalmodels. Intravenous infusions are appealing because of theirpracticality, low cost and applicability to a broad group of patients.Furthermore, treatment can be easily repeated and may not require anyspecial facilities. The downside includes systemic exposure to a growthfactor and the potential for adverse effects such as NO-mediatedhypotension.

[0035] Intracoronary infusions are easily carried out in any cardiaccatheterization laboratory and are also applicable in most patients withcoronary disease. However, the need for left heart catheterizationlimits this approach to a single session or, at most, infrequentrepetitions. While somewhat more “local” than intravenous infusions,intracoronary infusions are also likely to result in systemic exposureto the growth factor and may precipitate systemic hypotension. Avariation on the same theme is transvascular intracoronaryadministration with a local delivery catheter. This approach, while itis potentially feasible, remains experimental at this time, and it isstill associated with significant systemic recirculation.

[0036] Detailed evaluation of tracer-labelled growth factor uptake andretention in the myocardium, and its systemic distribution followingintracoronary and intravenous infusions, demonstrated that both forms ofdelivery are associated with relatively low uptake in the target(ischemic) area of the heart. Thus, at 1 hour after injection, 0.9% ofthe injected bFGF was found to be present in the ischemic myocardiumfollowing intracoronary administration and 0.26% following intravenousadministration. Perhaps more importantly, 24 hours later, very smallamounts of the growth factor remained in the myocardium (0.05% forintracoronary and 0.04% for intravenous administration).

[0037] Intramyocardial delivery of growth factors is the least evaluatedform of therapy at this time. The appeal of this mode of deliveryincludes the possibility of targeting the desired areas of the heart,which is likely to provide higher efficiency of delivery and prolongedtissue retention. The drawbacks are its invasive nature, andrequirements for highly specialized equipment and a high skill level forthe operator. Furthermore, no conclusive data regarding thephysiological efficacy of this mode of administration are available todate.

[0038] The pharmacokinetics and tissue distribution of protein growthfactors administered by various techniques have not been clearlydefined. Although an i.v. delivery strategy is very appealing in termsof technical safety, ease of administration, and lack of need forcardiac catheterization, it is unclear whether i.v. delivered growthfactors achieve therapeutic myocardial concentrations without untowardsystemic effects. In addition, IC infusions may not result in moresignificant myocardial deposition and retention with the addedinvasiveness of the delivery technique. The relevance of tissuedistribution becomes apparent when one considers the potential systemictoxicity of these agents in terms of hemodynamic effects, recirculation,and organ deposition, with the potential to induce pathologicangiogenesis and tumor genesis.

[0039] Pulmonary Routes of Administration Pulmonary delivery ofpotentially therapeutic agents provides a direct route to thecirculation, with a minimum of discomfort and pain, and is acost-effective alternative in comparison to the more invasive routes ofdelivery typically utilized for administration of FGF, VEGF, and relatedproteins. Traditionally, noninvasive delivery systems do not work formacromolecules; pills or tablets enter the stomach, where enzymes andhydrochloric acid rapidly degrade the protein or peptide. The oraladministration of proteins and peptides is under investigation, but nosatisfactory system is commercially available yet. No acceptabletransdermal delivery systems have been found because of proteins' sizeconstraints or inherent physical properties that prohibit these largemolecules from crossing the diverse layers of the skin without theaddition of irritating enhancers.

[0040] The biology of the lung makes it a favorable environment fornoninvasive drug delivery (see FIG. 1). Studies have shown that mostlarge-molecule agents are absorbed naturally by the lungs, and onceabsorbed in the deep lung, they pass readily into the bloodstreamwithout the need for enhancers used by other noninvasive routes. Patton,J. S. Adv. Drug Delivery Rev. 1996, 19, 3. On inhalation, air passesthrough the trachea, which branches more than 17 times into successivelysmaller tubes that constitute the bronchial network, eventually reachingthe grapelike clusters of tiny air sacs known as alveoli. Each breath ofair is distributed deep into the lung tissue, to the alveolarepithelium, the surface area of which measures ^(˜)100 m² in adults—roughly equivalent to the surface area of a standard singles tenniscourt. This large area is made up of about half a billion alveoli, fromwhich oxygen passes into the bloodstream via an extensive capillarynetwork.

[0041] The potentially most significant barrier to the delivery ofcompounds via the lungs is the tightly packed, single-cell-thick layerknown as the pulmonary epithelium. In the lungs, the epithelium of theairway is very different from that of the alveolus. Thick, ciliated,mucus-covered cells line the surface of the airway, but the epithelialcell layer thins out as it reaches deeper into the lungs, until reachingthe tightly packed alveolar epithelium. Most researchers believe thatprotein absorption occurs in the alveoli, where the body absorbspeptides and proteins into the bloodstream by a natural process known astranscytosis.

[0042] Logically, there is no reason to expect safety problems relatedto the inhalation of a substance to be any different from thoseassociated with the injection of the same amount of the substance. Agrowing quantity of safety data indicates that inhaling proteins can besafe for patients with healthy or diseased lungs. The safety oftherapeutic inhalation is further supported by the existence of morethan 20 small-molecule and one large-protein drug inhalationproducts-approved by the U.S. Food and Drug Administration (FDA); thisgroup of therapeutic inhalants contains 13 different excipients.

[0043] Most aerosol systems today deliver a total amount of <100 μg ofdrug per puff to the deep lung; this amount is too low to enable timelydelivery of many macromolecules if the required dose is in the milligramdoses. Traditional inhalation systems have been designed primarily todeliver some of the most potent drugs in use today, the bronchodilatorsand bronchosteroids to treat asthma. Both types of compounds arebioactive in the lung at 5-20 μg per dose. In contrast, many peptide andprotein compounds need to be delivered to the deep lung at much largerdoses of 2-20 mg. Adjei, A. L.; Gupta, P. K. Inhalation Delivery ofTherapeutic Peptides and Proteins; Marcel Dekker: New York, 1997.

[0044] Bioavailability After the aerosolized drug reaches the deep lung,it must be absorbed with high enough bioavailability to make the systempractical. As early as 1925, insulin inhalation for the treatment ofdiabetes was shown to work in humans, but the bioavailability was low(<3%). More recently, several inhalation studies comparing insulinadministration by aerosol inhalation (using cumbersome devices) and bysubcutaneous injection for the reproducibility of dosing have shown thatthe variability in glucose response to the two methods was equivalent.Bioavailability in more recent studies with aerosol insulin was up to25%, supporting the use of such a method of administration. Laube, B.L.; Georgopolos, A.; Adams, G. K. J. Am. Med. Assoc. 1993, 269, 2106.Insulin administered by oral inhalation effectively normalized diabeticpatients' plasma glucose levels without adverse effects. Numerouspatents have issued, directed to methods, formulations and devices forthe oral administration of insulin via inhalation therapy. See, forexample, U.S. Pat. Nos. %, 952,008; 5,858,968; and 5,91 5,378, thedisclosures of which are hereby incorporated specifically by reference.

[0045] Bioavailability studies in humans of the aerosol administrationof lutenizing hormone-releasing hormone (LHRH), a decapeptide, and itsanalogues also have demonstrated that appropriate bioactive systemiclevels can be achieved to treat conditions such as endometriosis andprostate cancer. Thus, using delivery and formulation technologyavailable today, as would be recognized by one of skill in theappropriate art, it will be possible to deliver an effective amount ofFGF and/or VEGF, and related growth factor proteins, in the treatment ofchronic and acute heart disease.

[0046] The mechanism of macromolecule absorption in the deep lung isthought to occur via normal physiological processes that can deliveractive compounds with relatively high bioavailability without requiringthe addition of penetration enhancers. LHRH analogues (used in treatingosteoporosis), composed of 10 amino acids, can reach 95%bioavailability; however, interferon-α (used in treating hepatitis B andC), composed of 165 amino acids, attains 29% bioavailability. Somesmaller peptides such as glucagon (29 amino acids) and somatostatin (28amino acids) reach 1% bioavailability. The degree of bioavailability isthought to depend on the peptide or protein susceptibility to certainhydrolytic enzymes in the lung.

[0047] How a macromolecular drug is formulated also affects its deliveryto the deep lung. Many macromolecules are formulated as dry powdersbecause they are more stable as solids than as liquids. Compared withliquid aerosol particles, which are mostly water (97%), dry powderaerosol particles can carry 50-100% of the drug. In general, more puffswould be necessary to deliver the equivalent amount of drug to thealveolar epithelium from a liquid aerosol device. Liquid formulationsalso carry the risk of microbial growth; the risk of lung infections dueto bacterial and fungal contaminants is greatly reduced with dry powdersystems. By greatly lowering the possibility of microbial contamination,dry powder systems offer a safer technology.

[0048] In the liquid state, individual protein or peptide molecules areextremely mobile. When water is removed, macromolecules usually packtogether in an amorphous state, unlike the highly ordered packing thatoccurs in crystallization. When water is removed from proteins, theprotein molecules remain mobile and chemical stability stays low in theinitial amorphous powder that forms. When a critical amount of water hasbeen removed, a kind of molecular gridlock occurs, producing a greatlyincreased chemical stability called the “amorphous glass state.” In thisstate, previously mobile molecules slow down drastically. As long as theglass transition temperature of the powder is higher than anyenvironmental temperatures that may occur during normal human use, thepowder will remain in a glass state.

Systematic, Multi-Tiered Approach to the Use of Growth Factor Proteinsin the Treatment of Acute and Chronic Heart Disease

[0049] Of the various treatment modalities currently in use or underinvestigation for the delivery of therapeutically effective doses ofvarious growth factor proteins, a wide range of levels of invasivenessare involved. Obviously, intravenous administration is among the leastinvasive, but questions remain as to the ultimate delivery of theproteins to physiological sites at therapeutically effective levels.Next most invasive is intracoronary infusion through catheters. Althoughrequiring surgical intervention, the insertion and manipulation ofcatheters has seen increasingly widespread use in the treatment of thesymptoms of heart disease and a number of other clinical conditions.However, for most, if not all, cardiac patients, there is a very lowlevel of toleration of such catheterizations procedures, so that thepossibility of repeated deliver of growth factor proteins is extremelylimited.

[0050] Next on the relative scale of invasiveness is intrapericardialinjection of growth factors. Although requiring more substantivesurgical procedures, this technique can be utilized in conjunction withother surgical procedures such as coronary artery bypass surgery andwould, thus, not constitute an additional traumatic burden on thepatent. Alternatively, relatively minor incisions can be made in thechest wall to permit direct interpericardial delivery. Again, due to theinvasive nature of the procedures utilized in this manner of delivery,the realistic possibility of repeated administration via this route isvery low.

[0051] At the most invasive end of the spectrum is direct myocardialinjection of FGF and related proteins. This, of course, requires openheart surgery to achieve access to the delivery site. As such, thisapproach is feasible only when used in conjunction with surgicalintervention for other purposes, such CABG. Again, the major drawbackhere is that there is very little practical opportunity for repeateddelivery of the therapeutic protein.

[0052] At the opposite end of the invasiveness spectrum, intrapulmonaryinhalation therapy, preferably via dry powder formulations, offerssignificant advantages over previous delivery strategies. As discussedabove, formulation and delivery technology has reached a state where anumber of therapeutic macromolecules, including insulin, can now bedelivered consistently, and at clinically effective levels viainhalation therapy. An added advantage arising from the non-invasivenature of inhalation therapy is that it is particularly attractive inthe treatment of chronic heart conditions that require repeated dosingover longer time intervals. The non-invasive nature of the therapy alsoproves to be of significant advantage in the treatment of acute heartconditions such as the onset of a myocardial infarct. For patients knownto be at risk for such a cardiac event, it will be possible to carry arelatively compact dry powder inhalation device so that at the onset ofsymptoms, the patient can selfadminister a dose of growth factor thatmay prove to be significantly effective in reducing the damage inducedby the MI, and may eventually prove to constitute the difference betweenlife and death.

[0053] Recognizing the scope of therapies potentially available in thetreatment of both acute and chronic heart disease, it is therefore anaspect of the present invention to provide a systematic, multi-tieredtherapeutic approach to the administration of FGF, VEGF and relatedgrowth factor proteins. This approach must, of necessity, recognize therelative invasiveness of different treatment modalities, and thelikelihood of repeated recourse to such treatment procedures.

[0054] In implementing the rational, multi-tiered therapeutic approachof the present invention, it is recognized that differing approachesneed be taken with respect to chronic and acute conditions. In the caseof chronic conditions, the initial tier of therapeutic treatment is theadministration of therapeutic levels of FGF (acidic or basic), VEGF, orrelated growth factor proteins, either individually or in combination,via dry powder inhalation therapy. Ideally, this therapy should beutilized as soon as possible after the onset of acute symptoms. For thisform of delivery, repeated doses can be administered, at levels and atdosage ranges as set forth in the examples below.

[0055] Upon appropriate monitoring of the clinical effectiveness of theinitial tier of therapy, as disclosed herein, the health practitionercan assess the advisability of proceeding to the next tier ofinterventional therapy. As described above, the next most invasive levelof therapy would entail the intracoronary delivery, via catheter, oftherapeutic doses of one or more of the growth factor proteins. Asalluded to above, in the acute stage of heart disease, the healthpractitioner does not have the option of a great deal of time in whichto assess the success of alternative treatment options. Thus, theability to assess, short-term, the efficacy of a particular treatment isessential to formulating the overall therapeutic strategy. The methodsof the present invention, disclosed below, for assessing on a short termbasis the effectiveness of growth factor protein treatment are essentialto the rational, multi-tiered approach to the treatment of heart diseasedisclosed and claimed herein.

[0056] Upon assessment that the clinical effectiveness of intracoronarydelivery of the protein growth factor has not met the desiredtherapeutic goal, the health care provider must consider optionsinvolving far more invasive surgical intervention. Among these would bethe intra-pericardial injection of FGF, VEGF, and/or other relatedprotein growth factors. If the health care provider has reached thepoint in assessment of therapeutic options where coronary angioplasty(PTCA) or bypass surgery (CABG) is mandated, then the delivery of one ormore growth factor proteins becomes feasible. At this level of therapy,for patients whose condition does not require PTCA or CABG, but whoseresponse to previous levels of therapy has not been adequate, analternative option is to utilize a limited thoracotomy forintrapericardial delivery of the therapeutic protein(s).

[0057] At a final level of therapeutic intervention, FGF or otherprotein may be delivered by direct injection into the myocardium duringtransmyocardial laser revascularization, or during coronary bypasssurgery. At this level of treatment, it is also possible to implantslow-release beads comprising the therapeutic protein for both long- andshort-term benefit.

[0058] An additional aspect of the treatment of acute symptomaticconditions is that, unlike uncontrollable incidents arising fromunstable angina, acute anginal attacks, or onset of myocardial infarct,certain therapeutic procedure have the potential to create symptoms thatcan be alleviated through administration of FGF, VEGF, and/or relatedproteins. Specifically, reperfusion injury can occur during anyprocedure when blood flow is temporarily curtailed or restricted, uponreinstitution of full blood flow. Examples would be in the course ofthrombolytic therapy (such as the administration of recombinant tissueplasminogen activator), as well as in bypass surgery and angioplasty. Asthe data included herein demonstrate, the extent of reperfusion injurythat can result in such situations can be ameliorated throughadministration of FGF, VEGF, and/or related proteins prior toreinstatement of full blood flow. Thus, the rational, multi-tiertherapeutic approach for the treatment of acute conditions of thepresent invention can be modified to include the administration of theappropriate growth factor protein or mixtures thereof prior toinitiation of the procedure raising the risk of reperfusion injury.

[0059] The rational, multi-tier approach to treatment of heart diseasewith FGF, VEGF and/or related growth factor proteins can be adapted totreatments for chronic, as opposed to acute, conditions. The initialtier, as with acute conditions, is based on delivery of the therapeuticproteins via inhalation therapy, preferably using dry powderformulations. Thus, for patients exhibiting the symptoms of chronicischemic disease, initial treatment involves inhalation therapy with atherapeutically effective amount and formulation of FGF, VEGF, and/orrelated proteins according to a dose level and dosing regimen as setforth in the Examples below. Due to the long-term nature of suchconditions, the progress from less invasive to more invasive treatmentmodalities does not need to progress on a shortened time scale as is thecase for treatment of acute conditions. Thus, multiple administrationsof the protein(s) via inhalation therapy are possible, preferablyaccompanied by clinical evaluation of the effectiveness of previoustreatments. In this fashion, dose levels and/or dose schedules can beadjusted based upon the results of periodic clinical evaluation of thepresence of markers such as CPK-MB, as disclosed more fully below.

[0060] If the clinical evaluations do not reveal sufficient progress inamelioration of symptoms associated with the disease state, then thehealth care provider can move to the next tier, or level, of treatment,moving further along the spectrum of increasing invasiveness. Thus, thenext tier would involve intracoronary perfusion via catheter. After aperiod of monitoring of the therapeutic effectiveness of theintracoronary perfusion, the health care provider can assess whether itwill b necessary to move to the next, more invasive, tier of treatment.Assuming that the patient's condition has not responded to treatment todate, then it is likely that the health care provider will be forced toconsider more invasive surgical treatments such as bypass surgery orcoronary angioplasty. If clinical conditions dictate such an escalationof therapy, then the next tier of therapy, interpericordial injection ofthe growth factor protein(s) can be implemented in conjunction with thesurgery. Alternatively, for patients whose condition does not warrant,or cannot support, angioplasty or bypass surgery, a limited thoracotomymay be used to achieve interpericordial delivery of the protein(s).

[0061] If symptoms or clinical testing do not evidence sufficientprogress in treatment, then the health care provider may elect to movetherapy to the highest tier of invasiveness. Thus, intermyocardialdelivery of FGF, VEGF and/or related proteins may be achieved inconjunction with surgical procedures.

[0062] Integral with the rational, multi-tier approach of the methods ofthe present invention, is the use of a rapid, easily accomplishedclinical evaluation procedure designed to provide the health careprovider with an indication of the efficacy of growth factor therapy.Accordingly, the present invention provides an assay technique thatsatisfies this need. As one of skill in the relevant art wouldrecognize, the criteria used to diagnose myocardial infarction (MI) canbe of critical importance clinically. The most widely accepteddiagnostic criteria for MI are those of the World Health Organization,first proposed over 20 years ago. These criteria require the presence ofat least 2 of the following 3 criteria: (1) a history of ischemic-typechest discomfort; (2) evolutionary changes on serial electrocardiograms;and (3) a rise and fall in serum cardiac enzymes. Joint InternationalSociety and Federation of Cardiology/World Health Organization TaskForce “Nomenclature and criteria for diagnosis of ischemic heartdisease,” Circulation 59: 707-709 (1979). Of importance here the use ofcreatine kinase (CK) and the more myocardium specific MB isoenzyme,CK-MB, as markers for MI. Wagner, G. S. “Optimal use of serum enzymelevels in the diagnosis of acute myocardial infarction,” Arch Intern Med140: 33-38 (1982). Data compiled in conjunction with the large,multicenter Platelet Glycoprotein IIb/IIIa in Unstable Angina: ReceptorSuppression Using Integrilin Therapy (PURSUIT) trial suggest that smallCK-MB elevations represent clinically important evidence of myocardialnecrosis and should be considered sufficient cardiac-marker criteria fora diagnosis of MI in patients with acute coronary syndromes. Alexander,J. H., et al., “Association Between Minor Elevations of CreatineKinase-MB Level and Mortality in Patients With Acute Coronary SyndromesWithout ST-Segment Elevation,” JAMA 283: 347-353 (2000).

[0063] The conclusion to be drawn from this data is that monitoring ofthe level of CK-MB can provide useful information for the clinicalpractitioner is assessing the short-term efficacy of various levels oftreatment with FGF, VEGF and/or related growth factor proteins. Thus,the method of the present invention contemplates implementation of arational, multi-tier therapeutic treatment strategy for administrationof growth factor proteins in patients with chronic and/or acute heartdisease, preferably with periodic evaluation of CK-MB levels as a markerof the clinical efficacy of growth factor delivery, and as an indicatorof the need to consider escalation of therapy to the next most invasivetier of treatment.

[0064] As would be recognized by one of skill in the appropriate art,methods and materials for the clinical monitoring of CK-MB levels arecommercially available and are routinely practiced in the context ofhealth care institutions.

EXAMPLES

[0065] Specific examples of the present invention are illustrated in thefollowing Examples that are not to be construed as limiting of the scopeof the claimed invention.

Example 1 Intracoronary Injection of FGF-2 in the Treatment of SevereIschemic Heart Disease: A Maximally Tolerated Dose Study

[0066] Patient selection. The study was conducted at two centers, theBeth Israel Deaconess Medical Center (Boston, Mass.) and EmoryUniversity Hospital (Atlanta, Ga.), and patients were enrolled betweenDecember 1997 and July 1998. The study was approved by the InstitutionalReview Boards at both hospitals. The inclusion criteria selected forpatents with advanced CAD with inducible ischemia and who wereconsidered to be suboptimal candidates for either PTCA or CABG. Patientswere excluded from the study if they had any of the following criteria:uncompensated congestive heart failure or an ejection fraction <20%; amyocardial infarction within three months; new onset of angina orunstable angina within three weeks; PTCA, CABG, stroke or transientischemic attack within six months; uncontrolled hemodynamicallysignificant arrhythmias; critical valvular disease; restrictive orhypertrophic cardiomyopathy; arteriovenous malformations; proliferativeretinopathy, retinal vein occlusion, or macular edema; renalinsufficiency (creatinine clearance<80 ml/min by 24-h urine collection);vasculitis or chronic immunosuppressive therapy; or any malignancywithin the past 10 years (except for curatively treated nonmelanoma skincancer). Patients with diabetes mellitus were eligible if they had noproliferative retinopathy or severe nonproliferative retinopathy, and nomicroalbuminuria.

[0067] Patient population. Fifty-two patients met all eligibilitycriteria and received a single IC infusion of rFGF-2. The mean age was60.8±10.1 years (range 41 to 80) and 2 of 52 patients were women. Sixpatients (11%) had diabetes mellitus and 31 patients (60%) had elevatedcholesterol (serum cholesterol>200 mg/dl). Forty-three patients (83%)had a history of at least one prior CABG. The mean ejection fraction(evaluated by MR imaging) was 51.4±12.0% (range 20% to 73%). Sixty-ninepercent of patients had NYHA class II or III symptoms of congestiveheart failure.

[0068] Study design. This was an open-label interpatient dose escalationstudy. The initial dose of 0.33 g/kg was escalated over eight sequentialgroups to 48 g/kg IC. At least four patients were studied at each dose.If no patient experienced dose-limiting toxicity as defined by theprotocol within six days, the dose was escalated; if one patientexperienced dose-limiting toxicity, an additional four patients werestudied at that dose. The MTD was defined as the dose tolerated by 9 of10 patients.

[0069] Study procedures. After providing informed consent and meetingall eligibility criteria, patients underwent baseline evaluations thatincluded a complete medical history and physical examination, anophthalmologic examination with fundus photography read by a corelaboratory using the Early Treatment Diabetic Retinopathy score (ETDRS),an exercise tolerance test (ETT), a Seattle Angina Questionnaire (SAQ),and nuclear and MRI cardiac scans. Measurement of initial health statusallowed the use of change in scores, thus adjusting for differences inbaseline health. Self-administration was used instead of telephoneinterview to minimize data collection bias.

[0070] On day 1, patients underwent right and left heart catheterizationand coronary angiography. If the coronary anatomy was not amenable toPTCA or CABG, recombinant FGF-2 (rFGF-2, Chiron Corporation, Emeryville,Calif.) was infused with a Baxter pump through diagnostic catheters intotwo major conduits of myocardial blood supply over 20 min (10 min ineach vessel) with continuous monitoring of systemic blood pressure andright atrial and pulmonary capillary wedge pressures, and cardiacoutput. In occasional patients the entire dose was infused into a singlevessel that was believed to be the major source of blood supply. Priorto initiation of rFGF-2 infusion, normal saline was administeredintravenously (i.v.), if required, to ensure mean pulmonary capillarywedge pressure>12 mm Hg. Heparin (40 U/kg) was administered i.v. morethan 10 min before rFGF-2. The volume of infusion varied with dose andthe patient's weight, ranging from 10 ml at lower does to 40 ml athigher doses.

[0071] The right heart (Swan-Ganz) catheter was left in place for 7 hfollowing drug infusion to monitor filling pressures and cardiac output.Patients were monitored with full-disclosure telemetry for 24 hfollowing rFGF-2 administration. Patients were discharged 24 h afterstudy drug infusion and clinical follow-up visits were performed at days6, 15, 29, 57, 180 and 360. Quality of life was assessed using theSeattle Angina Questionnaire at baseline and days 57 and 180. ETT's wereobtained at days 29, 57 and 180. Exercise stressed nuclear perfusionscans (rest thallium/stress ^(99m) Tc-sestamibi) and resting cardiacmagnetic resonance scans were performed at days 29, 57 and 180.

[0072] Preliminary Efficacy of RFGF-2 Therapy. Although the small samplesize and the absence of a control group preclude any definitiveconclusions regarding efficacy, several findings suggest potentialclinical benefits of intracoronary rFGF-2 administration. In particular,quality of life, as assessed by the SAQ, improved in treated patients atday 57 compared with baseline, and this improvement was sustained forsix months. The magnitude of improvements in the five SAQ scales wassimilar to that seen following PTCA and CABG in patients with ischemicheart disease. There was also a significant improvement in exercisecapacity, as measured by exercise treadmill testing, seen at days 57 and180. Of note, there was minimal improvement at day 29. The lateoccurrence of improvement in exercise testing is in keeping with theassumed time course of coronary angiogenesis. However, the absence of adose response tempers the preliminary efficacy seen in this study.

[0073] In addition, to these subjective measures of clinical status,resting MR imaging was performed to assess left ventricular function andmyocardial perfusion. Using this approach, we detected no difference inoverall left ventricular ejection fraction at any time during the study.However, there was a significant improvement in systolic thickening ofthe target wall at day 29, which was maintained at six months, and wasparalleled by a significant reduction in the size of the ischemicmyocardium as assessed by blood arrival imaging. Although cardiac MRimaging is considered the “gold standard” for evaluation of leftventricular function, its application to clinical trials in coronarydisease is very limited. Similarly, despite recent advances in MR-basedperfusion assessment of the myocardium, there has been no substantialclinical experience with this imaging modality. Prior animal studieshave documented improvement in MR-assessed parameters of leftventricular function in the setting of angiogenic growth factor therapy.In addition, the newly developed variation of MR perfusion imaging thatrelies on generation of space-time maps proved capable of detectingchanges in coronary perfusion in a pig ameroid model and proved capableof detecting improved regional myocardial perfusion in patients treatedwith epicardially administered sustained release FGF-2.

[0074] A fundamental question pertaining to IC delivery is how a drugwith a relatively short plasma half-life can promote a relativelylong-term process such as new collateral formation. One possibleexplanation is that first-pass extraction at the desired site of actionis the primary determinant of FGF-2 biological effect. Although suchextraction certainly occurs, animal studies demonstrated that <1% of¹²⁵I-FGF-2 administered using the intracoronary route is deposited inthe myocardium at 1 h and much less remains at 24 h. Although there isenhanced first-pass FGF-2 uptake in ischemic compared with normalmyocardium, presumably due to increased expression of cellular heparinsulfates and FGF receptor-1, myocardial levels fall to very low levelsat 24 h in both normal and ischemic regions of the heart. Onespeculative explanation is that this transient accumulation of FGF-2 inthe ischemic myocardium sets in motion a self-amplifying cascade thatincludes the influx and endothelial adhesion of monocytes/macrophagesand stimulation of expression of VEGF and other angiogenic cytokines,which may lead to prolonged and sustained action.

[0075] Safety Assessment. The safety of intracoronary rFGF-2 wasassessed through clinical observations, electrocardiography, hemodynamicmonitoring, hematologic and serum chemistry profiles, development ofanti-rFGF-2 antibodies, detailed ophthalmological exams with fundusphotography and assessment of renal function by determination ofcreatinine clearance and proteinuria using 24-h urine collection.Dose-limiting toxicity was predefined as a persistent (>10 min) drop insystolic blood pressure by >50 mm Hg, change in heart rate to >120/minor to <50/min, new clinically significant arrhythmia, new ischemicsymptoms or ECG changes, new congestive heart failure, deterioration inrenal function or any other serious adverse events.

[0076] Clinical follow-up and safety assessment. Clinical follow-up ofat least six months was obtained on all patients. A total of 30 seriousadverse events were reported in 22 patients. There was no apparentrelationship between increasing dose of rFGF-2 and serious adverseevents.

[0077] Four patients died. Two deaths were sudden and occurred 22 days(0.65 g/kg dose, EF 30%) and 114 days (48 g/kg dose, EF 22%) afterrFGF-2 infusion. One death was due to complications of cardiactransplantation and one death was due to complications of large-celllymphoma. Both instances of sudden death occurred in patients withreduced left ventricular function (22% and 30%). Although sudden deathmay be part of the natural history of their disease, potential partialrevascularization in these patients may have induced ventriculartachyarrhythmias. The diagnosis of large-cell non-Hodgkin's lymphoma 10days after rFGF-2 infusion most likely reflected the presence of diseasethat antedated IC rFGF-2 administration. Nevertheless, it is possiblethat rFGF-2 may have exacerbated the lymphoma course.

[0078] One patient (2 g/kg) died 72 days after rFGF-2 infusion fromcomplications of cardiac transplantation after sustaining severalmyocardial infarctions beginning four days after drug infusion. Onepatient with preexisting lymphadenopathy (6 g/kg) died at 62 days fromseptic complications of large-cell lymphoma, which was diagnosed at 10days after dosing. In retrospect, the lymphoma most likely predatedrFGF-2 infusion. One additional patient was diagnosed with metastaticadenocarcinoma to the liver at day 431.

[0079] Four patients had non-Q-wave myocardial infarctions at days 5 (2g/kg dose group), 68 (6 g/kg), 132 (0.33 g/kg) and 146 (48 g/kg). Fourpatients had revascularization procedures (CABC and aortic valuereplacement in one patient at day 68 [6 g/kg] and PTCA in three patientsat day 100 (0.33 g/kg], 290 [24 g/kg], and 223 [48 g/kg]). One patientdeveloped atrial fibrillation at day 37. The most commonly reported(>10% of patients) adverse events were asthenia (19%), hypotension(15%), dyspnea (13%), insomnia (13%), angina (12%) and palpitations(12%). Of these asthenia, hypotension, insomnia, and dyspnea were morecommon at higher doses. No patients withdrew from the study because ofadverse events. Transient leukocytosis was observed in half the patientsat ≧24 g/kg. Fluctuations in renal function occurred but were transientand not dose related. Proteinuria (>250 mg/24 h) occurred in fourpatients (7.8%). Ophthalmological exams with fundus photography atbaseline and day 57 were available for 45 patients; seven patientslacked wither baseline or 57-day assessments. Forty patients (89%)showed no change from baseline, two patients improved by two ETDRSgrades and three patients worsened by two grades (0.65, 2.0 and 36.0g/kg groups).

[0080] Safety and Tolerability of RFGF-2 Administration. The ability toadminister fairly high does of rFGF-2 (up to 36 g/kg IC) withoutsignificant hemodynamic effects is somewhat surprising given priorreports of severe FGF-2-induced hypotension and the known capacity ofthis cytokine to stimulate NO release and induce arteriolarvasodilation. Hypotension was dose-related and dose limiting, but wasrapidly correctable by IV fluids. This finding is in sharp contrast toclinical experience with another NO-releasing growth factor, VeGF-A₁₆₅,where profound hypotension limits systemic administration. Thisdifference in part may be attributable to careful hemodynamic monitoringin these patients and a requirement for adequate pressure (>12 mm Hg)before initiation of rFGF-2 infusion.

[0081] Preclinical studies as well as limited clinical experience todate suggested that renal insufficiency due to membranous nephropathyaccompanied by proteinuria may be the most significant long-term sideeffect of FGF-2 administration. In this small trial, only four instancesof proteinuria were observed. In should be noted, however, that allpatients studied had normal renal function at baseline.

[0082] Additional serious side effects included the occurrence ofnon-Q-wave myocardial infarction in four patients, raising thepossibility that FGF-2 may have promoted growth, or destabilization ofcoronary plaque owing to its broad-spectrum mitogenicity and chemotacticactivity. The latter possibility may be particularly relevant given theability of FGFs to induce angiogenesis in vasa vasorum and theassociation between plaque angiogenesis and its growth and stability.Although these concerns are certainly worrisome, in the absence of acontrol group casual relationships cannot be confirmed or discounted.

[0083] Statistical methods. Data are pooled for all dose groups.Baseline characteristics and acute hemodynamic parameters are expressedas mean±standard deviation. Efficacy variables were analyzed using alinear mixed effects model with an unstructured covariance assumptionfor the repeated measurements, fit using the restricted maximumlikelihood method. Model-based estimates of the means±standard errors(SEM) are presented. An overall F-test for equality across all timepoints was conducted first. If this initial test was statisticallysignificant, pairwise t tests to compare baseline with each on-studytime point were performed at the nominal a-level. All reported p-valuesare two-sided, and a p-value<0.05 was considered statisticallysignificant.

[0084] Magnetic resonance (MR) imaging. Magnetic resonance (MR) imagingwas performed at baseline and days 29, 57 and 180 in the body coil of a1.5 T whole-body Siemens Vision or Philips NT system. Functional imagingwas performed during breath-hold using shared-center FLASH or multishotechoplanar imaging in each of the three mutually perpendicular standardviews, producing 16-24 sequential image frames each, collected overapproximately 12 heartbeats to measure regional wall systolicthickening. MR blood arrival imaging was assessed as previouslydescribed. A series of four inversion recovery images was obtained withthe inversion time (TI) adjusted to minimize signal intensity frommyocardium. Using the best TI for nulling myocardial signal, a series ofconcurrent parallel images were acquired in diastole during breathhold,at baseline and after the bolus injection of contrast media (0.05mmol/kg gadodiamide). Measurement of the timing of half-maximum signalarriving in the different parts of the myocardium demonstrated theexistence of several distinct regions, including normal myocardium andareas exhibiting delayed contrast arrival (ischemic zones). For eachscan, a pace-time map demonstrating distribution of contrast signaldensity over the left ventricular wall as a function of time wascreated. The extent of the territory demonstrating delayed arrival ofcontrast, defined as >1-s delay of contrast density reaching its 50%maximum value reflecting the most severely hypoperfused part of themyocardium, was then calculated and expressed ads percent of the totalleft ventricular myocardial area. MR analysis was performed by a corelab blinded to rFGF-2 dose assignment and to study sequence.

[0085] Quality of life assessment. There were significant improvementsin all five scales of the Seattle Angina Questionnaire at days 57 and180, as compared with baseline. In particular, angina frequency scoreincreased (denoting improvement) from 39.8±3.8 at baseline to 68.8±4.0(p<0.001) at day 57 and 64.7±4.5 at day 180 (p<0.001), overall p<0.001.Exertional capacity score increased from 49.2±2.8 at baseline to64.5±3.1 at day 57 (p<0.001) and 73.0±3.8 at day 180 (p<0.001), overallp<0.001.

[0086] Exercise treadmill testing. A subset of patients with matchingbaseline and follow-up exercise treadmill protocols was selected foranalysis. Among this group, the mean exercise time improved from 510±24s at baseline (n=35) to 561±26 s at day 29 (n=28; p=0.023), 609±26 s atday 57 (n=31; p<0.001), and 633±24 s at day 180 (n=23; p<0.001).

[0087] Left ventricular function assessment. Magnetic resonance imagingwas performed in 51 patients at baseline and was repeated at days 29(n=47), 57 (n=45) and 180 (n=31) to assess resting left ventricularejection fraction, regional wall motion, and myocardial contrastarrival. There was a small improvement in overall left ventricularejection fraction over the course of the study (baseline 51.4±1.7%, day29: 54.2±1.7% [p=0.02], day 57: 55.2±1.9% {p=0.003], day 180: 57.2±1.7%[p<0.001], overall p=0.002). The hypoperfused target area was selectedfor resting regional left ventricular wall motion analysis. Systolicthickening of this area (target wall) and normal wall were measuredusing a semiautomated quantification algorithm of short-axis MR images.Resting normal wall systolic thickening was 46.1±1.6% at baseline anddid not change significantly throughout the study duration (p=0.16).Resting target wall thickening was significantly lower than normal wallthickening at baseline (34.0±1.7% vs. 46.1±1.6%, p<0.001). Target wallthickening significantly improved at days 29, 57, and 180 as compared tobaseline [baseline: 34±1.7%, day 29: 38.7±1.9% (p=0.006), day 57:41.4±1.9% (p<0.001), and day 180: 42.0±2.3% (p<0.001), overall p=0.001].

[0088] Myocardial perfusion assessment. Myocardial perfusion wasassessed using MR imaging. The mean size of the delayed contrast arrivalzone was 15.4±0.8% of the left ventricle at baseline and was similar tothe global left ventricular extent of ischemia determined by nuclearperfusion imaging (17.3 ±1.8%). The size of the myocardial areademonstrating delayed contrast arrival was significantly reduced frombaseline (15.4±0.8%) at day 29 (9.0±0.6%, p<0.001), day 57 (5.6±0.7%,p<0.001) and day 180 (4.9±0.8%, p<0.001), overall p<0.001.

[0089] There was no correlation between the dose and the variousefficacy parameters studied.

Example 2 Safety and Efficacy of a Single Intrapericardial Injection ofFGF-2 In a Porcine Model of Chronic Myocardial Ischemia

[0090] Chronic Myocardial Ischemia Model. Yorkshire pigs of either sexweighing 15 to 18 kg (5-6 weeks old) were anesthetized withintramuscular (i.m.) ketamine (10 mg/kg) and halothane by inhalation. Aright popliteal cut-down was performed and a 4 French arterial catheterwas inserted for blood sampling and pressure monitoring. Leftthoracotomy was performed through the 4th intercostal space. Thepericardium was opened, and an ameroid constrictor of 2.5 mm i.d.(matched to the diameter of the artery) was placed around the leftcircumflex coronary artery (LCX). The pericardium was closed using 6-000Prolene suture, (J&J Ethicon, Cincinnati, Ohio) and the chest wasclosed. A single dose of i.v. cefazolin (70 mg/kg) was given, and i.m.narcotic analgesics were administered as needed. Animals then wereallowed to recover for 3 weeks (time sufficient for ameroid closure)before growth factor delivery. The treatment of animals was based on theNational Institutes of Health guidelines, and the protocol was approvedby the Institutional Animal Care and Utilization Committee of the BethIsrael Deaconess Medical Center.

[0091] Growth Factor Delivery. Three weeks after ameroid placement,animals were anesthetized with i.m. ketamine (10 mg/kg) and isofluraneby inhalation. A right femoral cut-down was performed and an 8 Frencharterial sheath was inserted for blood sampling, pressure monitoring,and left heart catheterization. Coronary angiography was then performedin multiple views using a 7 French JR4 diagnostic catheter (Cordis,Miami, Fla.) to confirm LCX occlusion and to assess the extent ofcollateral circulation in the LCX distribution (“collateral index”).After LCX occlusion was documented, percutaneous subxyphoid pericardialaccess was undertaken. With the animals in the supine position, theepigastric area was prepped and draped. An epidural introducer needle(Tuohy-17) was advanced gently under fluoroscopic guidance with acontinuous positive pressure of 20 to 30 mm Hg. Entry into thepericardial space was confirmed by the injection of 1 ml of dilutedcontrast. A soft floppy-tipped guidewire was then advanced into thepericardial space and the needle was exchanged for a 4 French infusioncatheter.

[0092] The animals were randomized to one of five treatment groups:

[0093] 1. Control: intrapericardial saline (n=10).

[0094] 2. Heparin: intrapericardial heparin (3 mg, n=9).

[0095] 3. FGF-2 30 μg: intrapericardial FGF-2 (30 μg)+3 mg of heparin(n=10).

[0096] 4. FGF-2 200 μg: intrapericardial FGF-2 (200 μg)+3 mg of heparin(n=10).

[0097] 5. FGF-2 2 mg: intrapericardial FGF-2 (2 mg)+3 mg of heparin(n=10).

[0098] The infusate was diluted to 10 ml with saline and infused over 5min with continuous electrocardiographic and pressure monitoring. Thecatheter was withdrawn, and a set of colored microspheres (blue) wasinjected into the left atrium to obtain baseline (pretreatment)myocardial blood flow. Finally, a magnetic resonance study was carriedout to obtain quantitative measures of global and regional leftventricular function [ejection fraction (EF) and radial wall motion] andassessment of perfusion using myocardial contrast density mapping. Theanimals then were allowed to recover for 4 weeks.

[0099] Final Study. Four weeks after intrapericardial agentadministration, all animals underwent final evaluation. Pigs wereanesthetized with i.m. ketamine (10 mg/kg) and isoflurane by inhalation.A left femoral cut-down was performed and an 8 French arterial sheathwas inserted for blood sampling, pressure monitoring, and left heartcatheterization. Coronary angiography was performed again in multipleviews. A second magnetic resonance study was carried out for global andregional left ventricular function and myocardial perfusion. Myocardialblood flow was determined using colored microspheres at rest (yellow)and after maximal coronary vasodilation with i.v. adenosine (white).Animals then were euthanized under anesthesia and the heart was obtainedfor additional analysis. In addition, a detailed macroscopic andhistologic postmortem examination was carried out on three animals ineach group.

[0100] A total of 56 animals survived ameroid placement around the LCXcoronary artery with resultant total LCX occlusion at 3 weeks. Sevenanimals died after being randomized to a treatment group. Six of theseseven animals died within 72 h of intrapericardial agent delivery. Ofthe seven animals deaths, two animals died of hypoxemia (one controlanimal and one FGF-2 30 μg animal) due to failure of mechanicalventilation before growth factor delivery, four animals died during MRI(three animals died before growth factor delivery and one afterpericardial access and delivery, with two animals randomized to the 200μg FGF-2 group and two animals in the control group), and one animaldied of unknown cause 26 days after growth factor delivery (heparingroup). The remaining 49 animals were randomized to each of fivetreatment groups with 10 animals in each of the FGF-2 and saline controlgroups and 9 animals in the heparin group. There were no significanthemodynamic effects of intrapericardial FGF-2 administration at anydose; no changes in blood pressure, heart rate, or left atrial pressurewere observed with drug administration.

[0101] Angiographic Analysis. Coronary angiography was performed inmultiple views (right anterior oblique, anteroposterior, and leftanterior oblique views for the left coronary artery; right anterioroblique and left anterior oblique for the right coronary artery).Evaluation of angiographic collateral density was performed by twoindependent angiographers blinded to treatment assignment. Differencesin interpretations were resolved by a third angiographer. The collateralindex was assessed for left-to-left and right-to-left collaterals usinga 4-point scale (0, no visible collateral vessels; 1, faint filling ofside branches of the main epicardial vessel without filling the mainvessel; 2, partial filling of the main epicardial vessel; and 3,complete filling of the main vessel).

[0102] Coronary Angiography Baseline right and left coronary angiographywas available on all 49 animals and final angiography was available on47 animals. Left-to-left collaterals and right-to-left collaterals weremeasured (collateral index). The extent of left-to-left collaterals pre-(3 weeks after ameroid placement) and post-treatment (7 weeks afterameroid placement) in all groups shows a significant improvement overbaseline in the collateral index of all three FGF-2 treatment groups (30μg, 200 μg, and 2 mg) with no significant improvement noted in controlor heparin-treated animals. Only animals in the FGF-2 2 mg groupdisplayed a trend toward improvement in right-to-left collateral index(collateral index increased by 0.67±0.87, P=0.06).

[0103] Myocardial Blood Flow. Colored microspheres (15±0.1 μm diameter;Triton Technology Inc., San Diego, Calif.) were used to determinecoronary blood flow before treatment initiation (blue) and at the timeof final study (yellow and white). For determination of coronary flow at3 and 7 weeks after ameroid placement, an angiographic JR4 catheter wasadvanced into the left ventricle and manipulated to engage the leftatrium outflow by slow counterclockwise rotation of the catheter;catheter position was verified by contrast injection into the leftatrium. In addition, mean left atrial pressure was recorded. A set ofmicrospheres (6×106) was diluted in 10 ml of saline and injected intothe left atrium over 30 s. Reference blood samples were withdrawn byusing a syringe pump at a constant rate of 5 ml/min through the femoralartery. At the time of final study, coronary flow was measured at restand after maximal vasodilation (achieved with the injection of i.v.adenosine, 1.25 mg/kg). After study completion, the heart was excisedand regional myocardial blood flow was determined. The heart was excisedand a 1-cm midtransverse slice was sectioned and cut into eightsegments. The tissue samples and the reference blood samples weredigested in an 8 M KOH/2% Tween 80 solution and microspheres werecollected using a vacuum filter. Dyes from microspheres were extractedusing dimethyl formamide. Samples were then analyzed in aspectrophotometer (HP 8452 A; Hewlett Packard, Palo Alto, Calif.).

[0104] b. Analysis of regional wall motion using percentage of wallthickening.

[0105] c. Determination of the extent of coronary perfusion in the LCXcollateral-dependent territory compared with normal myocardium bymeasuring gadodiamide-enhanced signal intensity in different parts ofthe left ventricular wall and generating a space-time map of myocardialperfusion). The space-time maps allow the measurement of the extent ofthe ischemic zone.

[0106] MRI was available on 44 animals (8 in the control group; 9 in theheparin group; and 9 in each of the 30 μg, 200 μg, and 2 mg FGF-2groups). In five animals, MRI was not performed due to temporarytechnical problems with the MRI system at the time of the final study.The porcine ameroid occlusion model is associated with the developmentof small areas of left ventricular myocardial necrosis in most animals.

[0107] Global Left Ventricular Function. To assess the functionalsignificance of FGF-2-mediated improvement in myocardial blood flow, MRIwas used to assess global and regional left ventricular function in allstudy animals. There were no significant differences in global leftventricular function among the five groups (EF was 44.1±6.4% in controlsand 44.2±6.8% in heparin-treated animals versus 47.07±2.68 in the 30μgFGF-2 group, 45.52±3.41 in the 200 mg FGF-2 group, and 47.98±3.14 in the2 mg FGF-2 group; ANOVA, P =.35).

[0108] Regional Left Ventricular Function. Measurement of regional wallthickening in the LAD (normal territory) and LCX (ischemic) territorieswas used to assess regional left ventricular function (FIG. 2). LAD(normal) wall thickening was similar in all groups (ANOVA, P=0.86).FGF-2-treated animals had improved regional wall thickening in the LCX(ischemic) territory compared with controls and heparin-treated animals[FIG. 2; LCX wall thickening (%): controls, 33.58±9.91; heparin,32.64±13.45 (P=0.87 compared with controls); FGF-2 30 μg, 42.12±6.43(P=0.05 compared with controls); FGF-2 200 μg, 43.23±6.41 (P=0.03compared with controls); and FGF-2 2 mg, 47.14±3.64 (P=0.002 comparedwith controls); ANOVA, P=0.003]. Linear regression (assuming heparinresults in no significant FGF-2 release) revealed a dose-dependentimprovement in LCX wall thickening in the FGF-2-treated animals(y=37.6±0.005x, P=0.007)

[0109] Myocardial Perfusion. First-pass inversion-recovery turboFLASHMRI was used to generate a space-time map of myocardial perfusion (FIG.3 top). Three distinct zones are observed that are characterized byeither prompt signal appearance, failure of the signal to increase inintensity (infarction), or delayed signal appearance (delayed contrastarrival or ischemic zone). On the basis of contrast density data, atwo-dimensional map of contrast intensity versus time was generated andwas used to measure the size of the myocardial segments showing impaired(delayed) contrast arrival. FIG. 3 (bottom) depicts the extent of theischemic zone of contrast in the five groups. FGF-2 induced adose-dependent reduction in the extent of the ischemic zone, indicatingachievement of better myocardial perfusion in the FGF-2 treatment groups[FIG. 3 bottom; ischemic zone (% of left ventricle): controls,23.54±2.84; heparin, 22.41±6.85 (P=0.66 compared with controls); FGF-230 μg, 12.27±5.82 (P=0.0001 compared with controls); FGF-2 200 μg,6.63±1.97 (P <0.0001 compared with controls); and FGF-2 2 mg, 2.02±1.83(P <0.0001 compared with controls); ANOVA, P<0.0001; linear regressiony=16.7−0.008x, P<0.00011].

[0110] Histopathologic Analysis and Toxicology. Complete autopsies wereperformed on 15 animals (3 animals in each group). Tissues obtained fromthe liver, lung, kidney, spleen, eye, bone marrow, and stomach wereformalin-fixed and paraffin-embedded. Sections (5 μm) were obtained fromall tissue samples, stained with hematoxylin/eosin, and examinedmicroscopically. In addition, tissue samples were obtained frompericardium, epicardial coronary artery, and myocardium in the leftanterior descending coronary artery (LAD) distribution (normal) and LCXdistribution (ischemic). Sections were stained with hematoxylin/eosin aswell as by the Verhoeff-Van Gieson method for collagen and elastin.Complete serum chemistry and hematology studies were performed at 3 and7 weeks in all animals.

[0111] There were no treatment-related macroscopic or microscopicfindings in any of the organs examined. One animal had a single kidneypresent. there was focal to diffuse minimal thickening of thepericardium in all FGF-2 treatment groups, which was due to a slightincrease in connective tissue (fibrosis). There were minimal to mildchronic inflammatory cell infiltrates accompanied by focal or multifocalmineralization in all FGF-2 treatment groups. Increased vascularity wasnoted in the pericardium of two of three animals examined in the 200 μgFGF-2 group and one of three animals examined in the 2 mg FGF-2 group,but was not observed in the control, heparin, or 30 μg FGF-2 groups(FIG. 4B). In addition, the LAD and LCX in these animals were examinedand they showed no evidence of intimal hyperplasia.

[0112] Finally, there was an increase in vascularity of the epicardiumand myocardium in all animals from the 30 μg, 200 μg, and 2 mg FGF-2groups, but not in controls or heparin-treated animals. Sections fromthe LCX but not the LAD distribution in all FGF-2 treatment groupsshowed an increase in the number of capillaries. Many of these smallblood vessels were lined by endothelial cells that had largehyperchromatic nuclei, suggestive of new vascular in-growth (FIG. 4A).FGF-2 treatment did not result in any significant abnormalities in serumchemistries, hematology, and coagulation studies.

Example 3 Nonmitogenic Effects of Administration of FGF-2 in AcuteMyocardial Ischemia and Reperfusion in a Murine Model

[0113] To determine whether nonmitogenic effects of FGF-2 could bebeneficial to the heart during acute myocardial ischemia andreperfusion, FGF-2 was administered in a murine model of myocardialstunning. The advantages of this mouse model are well-defined markers ofischemia-reperfusion injury, including ischemic contracture, alterationin calcium homeostasis, and prolonged ventricular dysfunction, occurringwithin a time window too short to activate the mitogenic properties ofFGF-2. Transgenic mouse hearts deficient in the expression of theinducible isoform of NOS (NOS2−/−) were used to further investigate thecoupling of FGF-2 and NO during acute myocardial ischemia andreperfusion.

[0114] Stunning Myocardial stunning is the phenomenon whereby anischemic insult interferes with normal cardiac function, cellularprocesses, and ultrastructure for prolonged periods. Numerous mechanismsof myocardial stunning have been proposed, the most probable of whichinclude generation of oxygen-derived free radicals, metabolicimpairment, and calcium overload. Recently, a number of pharmacologicalagents and physiological manipulations have been shown to induce earlyor late ischemic preconditioning, a state characterized by reducedsusceptibility to postischemic decline in myocardial function. Inparticular, FGF-2 has been demonstrated to improve myocardial functionin the setting of acute myocardial ischemia both in vivo and in isolatedrat heart studies. The well-known angiogenic effects of FGF-2, however,occur too gradually to be relevant in such settings. The purpose of thisstudy, therefore, was to study the potential role of NO release inFGF-2-mediated cardioprotection and to define the NOS isoformresponsible for FGF-2-induced NO release.

[0115] Fifteen minutes of global ischemia followed by twenty minutes ofreperfusion resulted in prolonged ventricular dysfunction characterizedby reduced levels of LVP generation as well as significant decreases indP/dt_(max) and dP/dt_(min). Pretreatment with rFGF-2 significantlyimproved the extent of recovery of LVP compared with control (untreated)hearts (83±5 vs. 61±6%) and equally significant preservation ofdP/dt_(max) and dP/dt_(min) (86±3 vs. 65±6% and 85±5 vs. 60±5%,respectively. Stunning in hearts perfused with either NOS inhibitor byitself was not different from that in control hearts. Functionalrecovery of LVP in untreated control hearts (61±6%) was notsignificantly different from that in hearts perfused with either L-NAMEalone (59±9%) or L-NIL alone (57±6%). Depression of dP/dt_(max) anddP/dt_(min) (65±6 and 60±5%, respectively) in untreated hearts wassimilar to that in hearts perfused with L-abrupt and sustained rise inintraventricular pressure above 4 mmHg. Contracture time was measured asthe time from the onset of ischemia to the onset of contracture. At theend of 15 min of ischemia, the nitrogen-saturated bath was replaced bythe original bath maintained at 30° C. Flow was recommenced. Mean Ca_(i)²⁺ during early reflow was calculated as the mean of the peaks of Ca_(i)²⁺ recorded during the 1st minute of reperfusion. After 20 min ofreperfusion, Ca_(i) ²⁺ and functional parameters were again measured.

[0116] Drugs. Recombinant bovine FGF-2 (rFGF-2) was obtained from Chiron(Sunnyvale, Calif.). N-nitro-L-arginine methyl ester (L-NAME), aninhibitor of NOS, was obtained from RBI (Natick, Mass.).L-N⁶-(1-iminoethyl)lysine (L-NIL), a selective inhibitor of NOS2, wasobtained from Sigma (St. Louis, Mo.). All studies were conducted at 30°C., and hearts were paced at 6 Hz to minimize consumption of aequorin.After a 15-min equilibrium period, baseline conditions were recorded.Subsequently, hearts were divided into the following perfusion groups:perfusion with KH for 40 min (control, n=10), perfusion with KH for 20min followed by perfusion with KH plus 1 μg/ml rFGF-2 for 20 min(rFGF-2, n=10), perfusion with KH plus 400 μM L-NAME for 20 min followedby perfusion with KH plus 400 μM L-NAME plus 1 μg/ml rFGF-2 for 20 min(L-NAME +rFGF-2, n=6), and perfusion with KH plus 400 μM L-NIL for 20min followed by perfusion with KH plus 400 μM L-NIL plus 1 μg/ml rFGF-2for 20 min (L-NIL+rFGF-2, n=5). To test the effect of perfusion with theNOS inhibitors in the absence of rFGF-2, the following two additionalperfusion groups were studied: perfusion with KH for 20 min followed byperfusion with KH plus 400 μM L-NAME for 20 min (L-NAME, n=5), andperfusion with KH for 20 min followed by perfusion with KH plus 400 μML-NIL for 20 min (L-NIL, n=5).

[0117] Measurement of Intracellular Ca²⁺ In hearts in whichintracellular Ca²⁺ (Ca²⁺) was estimated, aequorin was injected into theapex of the heart. Briefly, after the perfusate was modified to contain0.5 mM CaCl₂, 0.6 mM MgCl₂, and 20 mM dextrose, 1-3 μl of aequorin wereinjected with a glass micropipette into a localized region of 2 mm² atthe apex of the heart. The heart was positioned in an organ bath suchthat the aequorin-loaded region was ^(˜)2 mm from the bottom of thebath. The Ca²⁺ and Mg²⁺ concentrations of the perfusate were increasedto 2.5 mM Ca²⁺ and 1.2 mM Mg²⁺ in a stepwise fashion over a period of 40min. The entire isolated heart preparation was positioned in alight-tight box for collection of the aequorin light signal. Aequorinluminescence was detected by a photomultiplier tube and recorded asanodal current. For estimation of Ca_(i) ²⁺, Triton X-100 was injectedinto the coronary perfusate to quickly permeabilize the myocardial cellmembranes and expose the remaining active aequorin to saturating Ca²⁺.This resulted in a burst of light, the integral of which approximatedthe maximum light (L_(max) ) against which light signals of interest (L)provided the fractional luminescence (L/L_(max) ). L/L_(max) wasreferred to a calibration equation to estimate Ca_(i) ²⁺.

[0118] Myocardial Calcium Homeostasis. Changes in myocardial Ca_(i) ²⁺are thought to play an important role in ischemia-induced myocardialdysfunction. Therefore, additional experiments were carried out toassess the effect of rFGF-2 administration on myocardial ionized calciumlevels. Myocardial Ca²⁺ measured at baseline was not different betweenNOS2+/+ and NOS2−/−hearts, and pretreatment with rFGF-2 had no effect onthese levels. Interruption of coronary flow produced abrupt alterationsin Ca_(i) ²⁺ in all hearts, with a gradual rise in diastolic and peakCa_(i) ²⁺ as ischemia progressed. Mean ischemic Ca_(i) ²⁺, Ca_(i) ²⁺averaged from the 2nd through the 14th minute of ischemia, was notaffected by rFGF-2 pretreatment and was the same in NOS2+/+ andNOS2−/−hearts. Restoration of coronary flow was followed by a markedincrease in myocardial Ca_(i) ²⁺. Neither the extent of this increasenor peak Ca_(i) ²⁺ levels was affected by rFGF-2 administration inNOS2+/+ or NOS2−/− hearts.

[0119] Measurement of NO Additional NOS2+/+ (n=5) and NOS2−/−hearts(n=5) were used to measure NO concentration in the coronary effluentusing an amperometric sensor (ISO-NO, World Precision Instrument,Sarasota, FL). Briefly, after 20 min of perfusion with either vehicle or1 μg/ml rFGF-2, the electrode was positioned in the effluent to measurethe amount of NO released from the coronary sinus. Electrode calibrationwas performed before each experiment with NO generated from the reactionof S-nitroso-N-acetylpenicillamine (Sigma) with cupric sulfate (Sigma)and acidic solution.

[0120] Quantification of NOS Gene Expression To determine NOS2 and NOS3mRNA levels in FGF-2-treated compared with control hearts, 30 cycles ofRT-PCR were performed on equal amounts of total RNA from six control andsix rFGF-2-treated hearts using primers corresponding to human NOS3 andNOS2 sequences. For NOS3, primers were as follows: 5′ (sense),5′-CAGTGTCCAACATGCTGCTGGAAATTG-3′ (bases 1,050-1,076); antisense,5′-TAAAGGTCTTCTTGGTGATGCC-3′ (bases 1,511-1,535). For NOS2, primers wereas follows: 5′ (sense), 5′-GCCTCGCTCTGGAAAGA-3′ (bases 1,425-1,441);antisense, 5′-TCCATGCAGACAACCTT-3′ (bases 1,908-1,924).Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was amplified fromthe same amount of RNA at the same time to correct for variation betweendifferent samples. The PCR products, separated on 1% agarose gels, werescanned and quantitated using Image-Quant software (Molecular Dynamics).

[0121] For Northern analysis of NOS1 and NOS3 mRNA levels in hearts ofNOS2−/− and wild-type mice, total RNA was prepared from freshly excisedhearts, subjected to electrophoresis on 1% paraformaldehyde-agarose gel,transferred to the GeneScreen Plus membrane (Dupont), and probed withrandom-primed mouse NOS1 and NOS3 cDNA probes. GAPDH cDNA probe was usedto control for loading. Quantification was achieved using Image-Quantsoftware.

[0122] Role of NOS2 The studies suggested that the NOS2 isoform was theprimary NOS isoform responsible for FGF-2-induced preservation ofmyocardial function in this model. To further corroborate these results,the same studies were repeated in hearts from NOS2−/−mice using theirNOS2 +/+littermates as controls. As in the case of previous studies,ischemia in both NOS2 +/+ and NOS2−/−hearts was characterized by anabrupt fall in LV pressure, a gradual onset of ischemic contracture, andprolonged ventricular dysfunction throughout 20 min of reperfusion.rFGF-2 pretreatment prolonged T_(LVP10), reduced the onset ofcontracture, and improved LV recovery throughout reperfusion. However,in NOS2−/−hearts, rFGF-2 failed to provide any protective effectsagainst global ischemia and stunning as measured by changes in LVP,dP/dt_(max), and dP/dt_(min) after 20 min of reperfusion.

[0123] Release of NO and FGF-2 Effects on NOS Gene Expression Todirectly demonstrate the role of rFGF-2-induced NO release, theconcentration of NO in coronary effluent before and after rFGF-2administration was measured. NO concentration increased significantlyafter perfusion with rFGF-2 compared with measurements after perfusionwith vehicle (236±24 vs. 190±25 nM/g, P<0.05) in wild-type hearts. Incontrast, perfusion with rFGF-2 did not increase NO concentration inNOS2−/−hearts compared with NO values measured after perfusion withvehicle (170±24 vs. 154±46 nM/g, P=NS). To assess whether rFGF-2increased NO production by stimulating NOS enzyme or increasing its geneexpression, we carried out RT-PCR analysis of NOS2 and NOS3 mRNA levelsbefore and after 40 min of exposure to rFGF-2. No differences in eitherNOS2 or NOS3 levels were detected.

[0124] The “knockout” of the NOS2 gene may have affected expression ofNOS1 or NOS3 genes in these mice. To evaluate this possibility, weperformed Northern analysis of NOS1 and NOS3 gene expression in heartsfrom C57/BL6 NOS2+/+ and NOS2−/−mice. No significant changes inexpression of either gene compared with that in control mice weredetected.

[0125] Statistical Analysis Observations made before and after drugadministration were compared using Student's two-tailed paired t-test.Observations made before and after the ischemia-reperfusion protocolwithin a group were compared using Student's two-tailed paired t-test.Between-group comparisons were made using analysis of variance. When anoverall significance was observed, multiple comparisons were performedusing the Bonferroni-modified t-test. A value of P<0.05 was consideredsignificant. Data are expressed as means±SE.

[0126] A porcine ameroid model was chosen for preclinical testing ofdelivery strategies because of several unique aspects. First, theameroid occluder results in consistent and gradual occlusion of the LCX,resulting in minimal myocardial necrosis, but reduced regionalmyocardial function, which is detectable with various noninvasiveimaging modalities. Because an effect of estrogen on cardiacangiogenesis cannot be ruled out and synchronization of these studieswith the menstrual cycle is logistically impossible, females wereexcluded from this study. In a similar model in dogs, dailyintracoronary injections of FGF-2 also induced increased vascularity ofischemic myocardium. Although very encouraging, there are little dataconsidering the efficacy of single intravascular administration ofangiogenic growth factors.

[0127] MATERIALS AND METHODS Male Yorkshire pigs (n=57; Parsons, Hadley,Mass.) weighing 15 to 30 kg were used for this study. The chronicischemia model consisted of three phases as previously described [4, 7].In brief, for ameroid surgery and catheterization at 3 and 6 weeks, theanimals were anesthetized with Ketamine 20 mg/kg IM and pentothal 10mg/kg IV, intubated, mechanically ventilated, and further anesthetizedwith 1.5% to 2.5% isoflurane in room air. Postoperatively, all animalsreceived antibiotics and analgesics for 48 hours. Animal care wasperformed according to the National Institutes of Health's Guidelinesfor the Care and Use of Laboratory Animals, and the protocol wasapproved by the Institutional Animal Care Committee.

[0128] A plastic ameroid (inner diameter, 2 to 2.5 mm; ResearchInstruments, Escondido, Calif.) was placed on the proximal leftcircumflex artery (LCX) or a major side branch, through a left lateralfourth intercostal thoracotomy. Three weeks (second phase, midstudy)later, right and left coronary catheterization was performed through astandard femoral cut-down after systemic anticoagulation with Heparin100 U/kg. Intraarterial pressure and electrocardiogram were continuouslyrecorded. Selective left and right angiography (General Electric,Waukesha, Wis.; contrast: Renografin; Squibb Diagnostics, Princeton,N.J.) confirmed complete occlusion of the LCX and allowed assessment ofbaseline flow and the presence of collaterals in the LCX territory,according to the Rentrop scoring system from 0 to 3: 0 =none; 1 fillingof side branches of the LCX; 2=partial filling of the LCX main arteryvia collateral channels; 3=complete filling of the LCX. Angiographicanalysis was blinded to treatment. For regional blood flow measurements,colored microspheres were injected into the left atrium (see below).Directly after this, function, perfusion, and collateral sensitivemagnetic resonance imaging (MRI) was performed on all animals toquantify baseline regional cardiac function and perfusion before startof the treatment.

[0129] Pigs were then randomly assigned to one of the followingtreatments: 1) vehicle control; 2) 2 μg/kg rFGF-2 IV; 3) 6 μg/kg rFGF-2IV; 4) 2 μg/kg rFGF-2 IC; 5) 6 μg/kg rFGF-2 IC. Five minutes beforeFGF-2 administrations, heparin (70 U/kg, IV) was given. Bovinerecombinant FGF-2 (rFGF-2; Chiron Corporation, Emeryville, Calif.) wasdissolved and diluted in vehicle consisting of 10 mmol/L sodium citrate,10 mmol/L thioglycerol, 135 mmol/L sodium chloride, 100 mmol/L EDTA, pH5.0. The intracoronary FGF-2 was equally divided and infused into theright coronary artery (RCA) and the proximal LCX using a 3F Cordisinfusion catheter. Intravenous infusions were given through an ear vein.In short proximal LCX stumps, FGF-2 was delivered into the proximal partof the LAD. The vehicle control group consisted of animals that receivedintravenous vehicle (n=4) or intracoronary vehicle (n=4). Three weeksafter therapy (third phase, final study), repeat selective angiogramswere made and two sets of colored microspheres were injected into theleft atrium, one before (rest) and one after injection of Adenosine 1.25mg/kg IV (stress). Function and perfusion MRI was also repeated in allanimals. Finally, animals were euthanized and the hearts were excised.

[0130] Fifty-seven animals received an ameroid constrictor and 13animals died before initiation of treatment. Forty-four animals(control, n=10; FGF 2 ,μg/kg IV, n=9; FGF 6 μg/kg IV, n=9; FGF 2 μg/kgIC, n=8; and FGF 6 μg/kg IC, n=8) completed the entire study.

[0131] Regional blood flow For microspheres injection into the leftatrium, a 7F JL4 catheter was retrogradely advanced across the aorticand mitral valve into the left atrium. The left atrial position of thecatheter was confirmed by contrast injection and the presence of anatrial pressure waveform. At midstudy, and during the final study atrest and stress, 6×10⁶ microspheres (Dye Trac; Triton Technologies, SanDiego, Calif.) were injected according to a standard protocol Referenceblood samples were drawn simultaneously. At the end of the study (finalstudy), a mid papillary, 1-cm-thick cross section of left ventricle wastaken and divided into eight radial segments. The segment in the LCXterritory was further subdivided in an endocardial and epicardial piece.Tissue samples and reference blood samples were digested and themicrospheres retrieved according to the manufacturers protocol. Thesamples were analyzed with a spectrophotometer (SU 600; Beckman,Fullerton, Calif.). From the optical density (OD) measurements, themyocardial flow was calculated as blood flow: (tissue sample X;mL/min/g)=[withdrawal rate (mL/min)/weight (tissue sample X; g)]×[OD(tissue sample X)/OD (reference blood sample)], using the Excelworksheet and macros provided by the manufacturer.

[0132] Hemodynamic parameters Intravenous infusion caused a mild butsignificant decrease in blood pressure of 12.3±3.7 mm Hg (p=0.02) in theFGF 2 μg/kg IV group and 9.6±2.1 mm Hg (p=0.01) in the FGF 6 μg/kg IVgroup. After intracoronary infusion, the drop in blood pressure wassignificant only at 2 μg/kg with 10.0±2.2 mm Hg (p=0.04) and not at 6μg/kg (6.1±4.9 mm Hg, p=0.25). In all groups, heart rate decreasedmildly, ranging from 2 to 15 bpm, but was significant only in the FGF 2g/kg IV with 9±4 bpm (p=0.05) and 6μg/kg IC group with 18±6 bpm(p=0.03).

[0133] Coronary angiography Seven follow-up angiograms, two in thecontrol group, two in the FGF 2 μg/kg IV, one in the FGF 6 μg/kg IV, andtwo in the FGF 2 μg/kg IC group, were not available for analysis.Collateral index had improved significantly in the 6 μg/kg IV group andin both 2 and 6 μg/kg IC groups, whereas baseline collateral index wassimilar (p=0.119, Kruskal Wallis). For all groups pooled, collateralindex resulted from left-to-left collaterals (either LAD to LCX or LCXto LCX, n=37; p<0.001, McNemar test) and not from right-to-left (p=1.0),sugggesting a localized effect of intravascular drug delivery. However,changes were not significant in any subgroup.

[0134] Coronary blood flow. Baseline regional blood flow in the ischemic(LCX) and normal (LAD) territories was measured at rest andposttreatment (final study) at rest and stress (adenosine). Absoluteischemic flow (mL/min/g tissue) and the LCX/LAD flow ratio weredetermined. LAD flow at baseline, rest, and stress at the final studywere similar in the five groups (ANOVA, p=0.363, p=0.418, and p=0.331,respectively). Rest LAD flow did not change significantly over time(ANOVA, p=0.266). In addition, LCX coronary blood flow at baseline(before FGF2 infusion) was similar in all five groups (ANOVA, p=0.361).At the final study at rest, absolute LCX flow and the LCX/LAD ratio didnot change significantly. However, LCX flow at stress was significantlyhigher in the FGF 6 μg/kg IC group than in controls (ANOVA, p =0.039).

[0135] Myocardial MRI analysis. Arterial pulse-gated MRI was performedon anesthetized (1% to 2% isoflurane) and ventilated animals, in thebody coil of a 1.5-Tesla whole-body (Siemens, Munich Germany) Visionprototype. Baseline anatomic images were obtained by a turboFLASHtechnique to identify coordinates for apical four-chamber, two-chamber,and short-axis views. For function studies, 24 sequential image frameswere collected over 12 heartbeats during breath-hold using shared-centerturboFLASH in each of the three standard views. After detection of theoptimal inversion time (TI; typically 200 to 300 ms), a series of 32diastolic images were acquired in the double-oblique four-chamber viewduring breath-hold, while injecting 0.05 mmol/kg gadodiamide(T1-reducing contrast agent). The series of images was viewed as amovie, to locate the zone with impaired contrast arrival. The short axisat the center of that zone (target zone) was prescribed graphically. Allmeasurements were performed by two independent investigators blinded totreatment. Custom-designed software was used to define myocardialborders and measure wall thickness. End-systolic and end-diastolic leftventricular volumes were computed from biplane measurement (apicalfour-chamber and two-chamber views) as previously validated, and used tocalculate left ventricular ejection fraction. Target wall motion (radialshortening) and target wall thickening were expressed as percentage ofthe radial length or wall thickness at the end of diastole. Bothparameters were also measured at the septum, yielding normal target wallmotion and target wall thickening. The area of delayed contrast arrivalwas defined as myocardium demonstrating distinctly slowed time (≧1cardiac cycle) to half-maximal signal intensity, using a two-dimensionalmap of contrast intensity versus time.

[0136] MRI: left ventricular function Infarct size visualized asmyocardium without MRI contrast uptake was measured to avoid confoundingof regional function and perfusion measurements. Infarct size wassimilar among the five groups at either baseline (3 weeks) or finalstudy (ANOVA, p=0.594 and p=0.303, respectively). Infarct size,3.0%±4.9% left ventricular area (mean±SD), was within the range reportedfor this model.

[0137] Left ventricular ejection fraction (EF) at baseline was similarfor all treatment groups (ANOVA, p 0.120). Using each animal as its owncontrol, EF improved significantly in controls (p=0.018), in the FGF 2μg/kg IV (p=0.046), the FGF 6μg/kg IV (p=0.001), and the FGF 6 μg/kg ICgroups (p=0.001). The improvement in EF after treatment wassignificantly higher in the FGF 6 μg/kg IC (p<0.01) group compared withcontrols. The improvement in indexed target wall motion (target wallmotion/normal wall motion) was significant only in the FGF 6 μg/kg IV(p=0.019) and the FGF 6 μg/kg IC groups (p=0.004), whereas indexedtarget wall thickening improved in the FGF 6 μg/kg IC group (ANOVA,p=0.007 compared with improved target wall thickening in controls,p=0.001).

[0138] MRI: perfusion. At baseline, no differences in areas of delayedarrival (ANOVA,p=0.140) or collateral extent (p=0.103) were foundbetween the groups. The size of the zone of delayed arrival decreased inthe FGF 6 μg/kg IC (p<0.001), which was significantly different from thechange in controls (ANOVA,p<0.001).

[0139] Toxicologic assessment of FGF-2 administration. Before treatmentand at necropsy, blood samples for hematology, coagulation, and serumchemistry were obtained from at least three fasted animals per group.Hematology parameters included hemoglobin, mean corpuscular hemoglobinconcentration, hematocrit, erythrocyte count, total leukocyte count,differential, platelet count, mean corpuscular hemoglobin, and meancorpuscular volume. Serum chemistry included aspartate aminotransferase,alanine aminotransferase, gamma glutyltransferase, alkaline phosphatase,lactate dehydrogenase, total bilirubin, total cholesterol,triglycerides, blood urea nitrogen, creatinine, creatine phosphokinase,albumin, globulin, total protein, electrolytes (Na, K, and Cl), calcium,phosphorus, and glucose.

[0140] In addition, for four randomly selected animals in each treatment(not vehicle) group, tissue samples were taken from major organs andprocessed for histology. Histopathological findings were graded on ascale of 1 to 4 (minimal<mild<moderate<marked), by a veterinarypathologist blinded to treatment.

[0141] There were no macroscopic or microscopic lesions related tointravenous or intracoronary administration of FGF-2. Furthermore, nochanges in hematological or biochemical parameters were observed in anyof the treatment groups.

[0142] In this study, in which the efficacy of intravenous andintracoronary delivery of 2 or 6 μg/kg FGF-2 was compared, blood supplyto the myocardium, as assessed by the colored microsphere method, wasimproved by the high-dose (6 μg/kg) intracoronary FGF-2. Although thiseffect was only significant at stress, the same trend was seen forregional blood flow at rest. Both intravenous FGF-2 doses as well as the2-μg/kg dose were ineffective. This change in regional blood flow wasconfirmed by perfusion and collateral-sensitive

[0143] It is concluded that a single 6-μg/kg intracoronary FGF-2delivery results in-significant improvement in collateralization andregional and global function of chronically ischemic myocardium. Asingle intravenous infusion of FGF-2 is ineffective in the doses tested.A phase 11 clinical trial of patients with coronary artery diseasedesigned to evaluate this intracoronary therapeutic strategy iscurrently underway.

Example 5 Local Perivascular Delivery of FGF-2

[0144] In this trial, patients with a viable and ischemic myocardialarea that could not be revascularized were randomized to receiveheparin-alginate pellets containing 10 or 100 μg of bFGF or placebo thatwere placed on the epicardial surface during CABG.

[0145] Patient Selection. The study population consisted of patientsundergoing CABG at Beth Israel Deaconess Medical Center and AlbertEinstein College of Medicine in Boston, Mass. The inclusion criteriaincluded an area of myocardium supplied by a major coronary artery withadvanced disease not amenable to bypass grafting or percutaneousintervention, inducible ischemia, and the ability to understand and signthe informed consent and to comply with planned follow-up. Patients withthe following criteria were excluded from consideration for the study:absence of inducible ischemia or myocardial viability of the targetarea, hypertrophic or restrictive cardiomyopathy, left ventricularejection fraction<20%, significant valvular heart disease, renaldysfunction (serum creatinine>2.5 mg/dL), history of malignancy withinthe previous 5 years, or unexplained hematological or chemicalabnormalities before CABG.

[0146] The design and performance of the study were approved by the Foodand Drug Administration under an investigator-sponsored investigationalnew drug (BB-IND 5725). The study was approved by the Committee forClinical Investigation at both institutions. The first patient wasenrolled in September 1996 and the last patient in May 1998.

[0147] Patient Population and Enrollment Procedure. Seventy-eightpatients scheduled for CABG were screened for enrollment into the studyon the basis of an angiogram that showed a major epicardial coronaryartery (posterior descending artery, significant diagonal, obtusemarginal, or ramus intermedius branch, or significant posterolateralbranch) that was considered by an interventional cardiologist and acardiothoracic surgeon not involved in the study unlikely to begraftable on the basis of its angiographic appearance (diffuselydiseased or heavily calcified). Patients were approached for enrollmentin the study, and screening tests were performed to ensure that alleligibility criteria were met, including demonstrable ischemia in thetarget myocardial area.

[0148] Forty-six patients who met all eligibility criteria and agreed toparticipate in the study underwent CABG, during which a noninvestigatorcardiac surgeon determined whether the target area was indeedungraftable. Bypass surgery of the target vessel was performed in 22cases, and those patients were excluded from additional study. Theremaining 24 patients (19 patients at Beth Israel Deaconess MedicalCenter and 5 at Montefiore Medical Center, Bronx, N.Y.) who had acoronary artery that could not receive a graft at the time of surgerywere randomized to receive 10 heparin-alginate pellets containingplacebo or 1 of 2 doses of bFGF (10 or 100 μg). There was no significantdifference between the study groups in any of the clinical parameters,including the extent of coronary disease or presence of any risk factor,except that patients in both 10-and 100-μg bFGF treatment groups weresomewhat older than controls, and there were more women in the 1 O-,μgbFGF group. The baseline resting ejection fraction was 50.3±13.8%, and 5of the 24 patients had an ejection fraction <30%.

[0149] Preparation of bFGF-Containing Heparin-Alginate Pellets. Calciumalginate pellets provide a stable platform for bFGF because of enhancedretention of activity and storage time and thus were used as devices forcontrolled bFGF release in vivo. Heparin-sepharose beads (Pharmacia LKB)were sterilized under ultraviolet light for 30 minutes and then mixedwith filter-sterilized sodium alginate. The mixed slurry was droppedthrough a needle into a beaker containing a hardened solution of CaCl₂(1.5% wt/vol). Beads formed instantaneously. Uniformly cross-linkedcapsule envelopes were obtained by incubating the capsules in the CaCl₂solution for 5 minutes under gentle mixing and then for 10 minuteswithout mixing. The beads were washed with sterile water and stored in0.9% NaCl-1 mmol/L CaCl₂ at 4° C. bFGF loading was performed byincubating 10 capsules in 0.9% NaCl-1 mmol/L CaCl₂-0.05% gelatin with12.5 p (for 10-μg dose) or 125 μ (for 100-μg dose) of bFGF (GMP gradehuman recombinant bFGF provided by Scios, Inc) for 16 hours under gentleagitation at 4° C. Previous studies have shown that under theseconditions, 80% of bFGF in solution is absorbed into heparin-alginatepellets. The end product was sterilized under ultraviolet light for 30minutes. With each preparation, several beads were cultured to ensuresterility. Blank or bFGF-loaded pellets were identical in appearance,which ensured that the surgeons and investigators were blinded withregard to which pellet was being used.

[0150] bFGF Heparin-Alginate Delivery After completion of coronarybypasses to all areas of the heart that could be revascularized andfailure to graft the target vessel (which on occasions involved probingof the target vessel), multiple linear incisions were made in theepicardial fat surrounding the target vessel. Heparin-alginate pellets(containing bFGF or placebo) were inserted into the epicardial fatoverlying the artery and secured in place by a 6.0 prolene suture toclose the subepicardial incision. A total of 10 pellets were used ineach patient (2 to 3 pellets were placed in each incision). The leftinternal mammary artery (LIMA) was placed on the left anteriordescending artery (LAD), and proximal vein-to-aorta anastomoses wereconstructed. Ventilation was reestablished, and cardiopulmonary bypasswas terminated. Routine closure was then performed.

[0151] Short-Term Results. The extent of CABG surgery was the same inall treatment groups; there were no significant differences with regardto the number of grafts, duration of surgery (average 3.0±0.9 hours), orcross-clamp time (average 56±13 minutes). The target vessel was theright coronary artery (RCA) in 15 patients, left circumflex artery in 7,and diagonal branch of the LAD in 2.

[0152] One patient in the control group died 24 hours after surgerysecondary to an autopsy-documented occlusion of one of the saphenousvein grafts, with a large myocardial infarction in that territory. Asecond death occurred in a patient in the 100-μg bFGF group who couldnot be weaned off cardiopulmonary bypass (preoperative ejection fractionof 20%); an autopsy revealed patent grafts with extensive myocardialscarring and a thin rim of epicardial viable myocardium. Two otherpatients (both in the control group) required intra-aortic balloon pumpsupport after surgery (in 1 patient, the intra-aortic balloon pump wasinserted before surgery). Two patients (1 in the control group and 1 inthe 10-μg bFGF group) had a Q-wave myocardial infarction in the targetmyocardial distribution, and 1 patient in the 10-μg bFGF group had aQ-wave myocardial infarction in a nontarget myocardial distribution.

[0153] Placement of bFGF-containing heparin-alginate microspheres had nosignificant short-term effects on blood pressure or heart rate; the meanarterial pressure was 84.8±10.6mm Hg before bypass, 89±12 mm Hg on day1, 93±7 mm Hg on day 3, and 83.4±11.1 mm Hg on day 5 and was notdifferent among the treatment groups. Pharmacokinetic evaluation did notreveal any significant increase in serum bFGF levels above baseline inany of the groups (average bFGF levels in 15 patients: 17.4±3.3,15.90±1.4, 15.9±1.8, and 16±1.8 μg/mL at baseline and postoperative days1, 3, and 5, respectively), and there were no significant differences inbFGF levels between the different treatment groups. The averagepostoperative hospital stay was 5.30±1.3 days (range 4 to 8 days). Therewere no acute effects on serum chemistries, hematologic and coagulationprofiles, liver function tests, or urinalysis. Two patients developedsuperficial wound infections along the chest incision that necessitatedsurgical debridement, and another patient with diabetes mellitus haddelayed healing of the saphenous vein graft harvest site.Microbiological evaluation of the beads showed no aerobic or anaerobicgrowth in samples from 28 of the 46 preparations.

[0154] In-Hospital Follow-Up. The postoperative course was evaluated,including-hemodynamic parameters, duration of ventilatory support,postoperative ECGs, postoperative cardiac isoenzymes, duration ofhospitalization, and any evidence of infection. Serum bFGF levels weremeasured (ELISA, R&D Systems) before implantation and on the first,third, and fifth postoperative days. Complete blood count, coagulationparameters, serum chemistries, and urinalysis were performed beforetreatment and at days 3 and 5 after treatment. In the first 10 patients,stress nuclear perfusion imaging and MRI (at the Beth Israel DeaconessMedical Center) were performed before CABG; however, owing to theconfounding effect of CABG (realized after an interim analysis of thefirst 10 patients by the Data Safety and Monitoring Committee), theremaining patients underwent stress nuclear perfusion scans(rest-thallium/dipyridamole sestamibi) and MRI after CABG (beforedischarge). The surgeon, other investigators, and patients were blindedto treatment assignment.

[0155] Long-Term Follow-Up. All patients were contacted by theinvestigators at 6 weeks; 2, 3, 4, and 6 months; 1 year; and then yearlythereafter to assess clinical events (death, myocardial infarction,recurrent angina, or any repeat revascularization). Complete bloodcount, coagulation parameters, serum chemistries, urinalysis, and serumbFGF level measurements were repeated at 3 months. Patients underwentstress nuclear scans at 3 months (dual-isotope studies with restthallium and stress [pharmacological stress with dipyridamolesestamibi]). In addition, patients at the Beth Israel Deaconess MedicalCenter underwent repeat MRI 3 months after CABG. Clinical follow-up of≧36 months was available for all patients, with a mean follow-up of16.0±6.8 months.

[0156] Clinical Follow-Up. Clinical follow-up was available in the 22surviving patients (7 from the placebo group, 8 from the 10 μg-bFGFgroup, and 7 from the 100 μg-bFGF group) and averaged 16.0±6.8 months.At last follow-up, all patients were angina-free except for 3 patientsin the placebo group (Canadian Cardiovascular Society [CCS] class II in1 and class III in 2 patients) and 1 patient in the 10-μg bFGF group(CCS class II). Two of the 3 placebo patients with angina underwentsuccessful percutaneous revascularization (1 involved the target vesseland the second involved a vein graft stenosis). After hospitaldischarge, none of the patients died or sustained a myocardialinfarction. There were no delayed wound infections, no clinical evidenceof pericarditis, and no other adverse events. Laboratory evaluation at90 days (available in 21 patients) did not show any adverse effect oncomplete blood count, coagulation parameters, serum chemistries, orurinalysis.

[0157] Imaging Studies. Rest thallium/dipyridamole sestamibi studieswere performed according to the ADAC protocol. We compared baseline and90-day nuclear scans using the size of the stress perfusion defect, asdetermined by pixel analysis. MRI was performed in the body coil of a1.5-T whole-body Siemens Vision system. Baseline anatomic images wereobtained by a turboFLASH (turbo Fast Low-Angle SHot) technique toidentify coordinates for apical 4-chamber, 2-chamber, and short-axisviews. Functional imaging was performed during breathhold by use ofshared-center turboFLASH in each of the 3 mutually perpendicularstandard views, producing 24 sequential image frames each, collectedover 12 heartbeats to measure regional wall motion. MR perfusion imagingwas performed as follows: a series of 4 inversion recovery images (1every second heartbeat) was obtained as inversion time (TI) and adjustedto minimize the signal intensity from myocardium in the fourth frame.With the best TI determined by these scout images, a series ofconcurrent parallel images were acquired in diastole during breathhold,1 every other heartbeat, at baseline and again with contrast injection(0.05 mmol/kg gadodiamide). In addition, complete blood count,coagulation parameters, serum chemistries, urinalysis, and serum bFGFlevel measurements were repeated at 3 months.

[0158] Nuclear Perfusion Imaging. Twenty of the surviving 22 patientsunderwent stress nuclear perfusion imaging 90 days after CABG. In thefirst 10 patients, baseline studies were performed before CABG. Itbecame clear as the study progressed, however, that this was not a truebaseline because of the confounding effect of CABG. Therefore, in theremaining 12 patients, rest-thallium/dipyridamole sestamibi nucleartesting was performed after CABG and before hospital discharge. Thebaseline stress target area defect size was 20.6±5.2% of the leftventricle and was similar in all 3 treatment groups (22.3±5.4% incontrols, 19.2±5.0% for the 10-μg bFGF group, and 20.4±5.7% for the100-μg bFGF group, ANOVA P=0.56). At the time of follow-up nuclearscans, when paired t tests were used, there was a trend toward worsening(increase in the defect size) in the placebo group (20.7±3.7% atbaseline to 23.8±5.7% at follow-up, P=0.06). Studies in the 10- μg bFGFgroup showed no change in defect size(19.2±5.0% to 16.9±8.1%, P=0.39),whereas defect size in the 100-μg bFGF group was significantly improvedcompared with baseline (19.2±5.0% to 9.1±5.9%, P=0.01). The change indefect size was significantly different among the 3 groups (ANOVAP=0.005). Semiquantitative analysis of stress images demonstratedworsening of the defect in 3 of 6 patients and no change in 3 of 6patients in the control group. Of 8 patients in the 10-μg bFGF group,the target nuclear defect size worsened in 2 patients, remainedunchanged in 2, and improved in 4. Finally, of the 6 patients in the100-μg bFGF group who underwent follow-up nuclear testing, there wasimprovement in 5 patients and no change in 1 patient.

[0159] Magnetic Resonance Imaging. Functional and perfusion MRI wereperformed in 8 patients at the Beth Israel Deaconess Medical Center atbaseline and at 90-day follow-up (4 controls and 4 bFGF-treated patients[1 patient in the 10-μg bFGF group and 3 in the 100-μg bFGF group]).Baseline resting target wall motion (radial wall motion) was 21.7±6.7%in the placebo group and 27.3±17.0% in patients treated with 100 μg ofbFGF (compared with 35.7±10.9% for normal revascularized wall). Nochanges in resting target wall motion were seen at follow-up (23.7±9.3%in placebo and 32.3±12.4% in 100-μg bFGF-treated subjects). The extentof the resting delayed contrast arrival zone, which reflectsunderperfused myocardium, for placebo and bFGF-treated patients was10.7±3.9% and 15.7±2.3% at baseline and decreased to 7.8±6.9% (P=0.37)and 3.7±6.3% (P=0.06) at follow-up, respectively, with a trend towardimprovement in the 100-μg bFGF group. of therapy in patients with viableand ischemic but unrevascularizable myocardium. These results warrant alarger multicenter trial to assess the clinical benefit of thiscombination approach to myocardial revascularization, which is currentlyunder way.

Example 6 Reduction in Myocardial Infarct Size Following IntracoronaryAdministration of FGF-2

[0160] The extent of myocardial injury and necrosis resulting from anischemic insult is determined by the duration of interruption toantegrade flow, the size of the compromised territory, and the extent ofcollateral circulation to the region. In view of the beneficial effectson myocardial viability and contractile function demonstrated incollateralized patients with occlusive coronary artery disease, thesefindings provide a rationale for investigation of new strategies thatuse growth factors such as bFGF to pharmacologically enhance collateralgrowth and to blunt the effects of impaired antegrade myocardialperfusion.

[0161] Coronary Occlusion and Reperfusion. Twenty-two mongrel dogs ofeither sex (weight, 17 to 23 kg) were randomly assigned to treatmentwith bFGF or vehicle. After the animals received anesthesia with sodiumpentobarbital (25 mg/kg IV), intubation, and ventilation with room air,the right carotid artery was exposed, ligated distally, and cannulated.Aortic blood pressure, heart rate, and ECG were monitored continuouslythroughout the procedure. After baseline left ventriculography wasaccomplished via a 6F pigtail catheter, selective left coronaryangiography was performed via an 8F angioplasty guiding catheter.Because of the potential interaction between heparin and bFGF,intraprocedural anticoagulation was achieved with the use of Hirulog, asynthetic direct thrombin inhibitor; after an intravenous loading doseof 2.5 mg/kg, intravenous infusion was commenced at 5 mg kg⁻¹h⁻¹ and therate adjusted to maintain the activated clotting time at >300 seconds.

[0162] An angioplasty balloon catheter (balloon:artery ratio 1.0) wasthen inflated at 2 atm in the middle part of the LAD distal to the firstdiagonal branch, and occlusion was confirmed angiographically. After 4hours' occlusion, the balloon catheter was deflated and removed, and LADpatency was confirmed angiographically. Ten micrograms of humanrecombinant bFGF (in 20 mmol/L sodium citrate, 1 mmol/L EDTA, and 9%sucrose, pH 5; Scios Nova Inc) in 10 mL normal saline or vehicle (10 mLnormal saline) was administered directly into the left main coronaryartery via the guiding catheter 10 minutes after occlusion and againjust before reperfusion. After reperfusion, left ventriculography wasrepeated. All surgical procedures were performed with the use of asterile technique. Seven days after the first procedure, dogs wereanesthetized, intubated, and ventilated in the same manner as before.Patency was confirmed angiographically, left ventriculography wasrepeated, and euthanasia was performed with a lethal dose ofpentobarbital.

[0163] Occlusion-Reperfusion Study. Blood pressure and heart rate weresimilar in both groups throughout the experiment. Heart rate wasincreased during reperfusion in vehicle- and in bFGF-treated dogs (bothP=0.043 versus baseline) because of nonsustained ventricular tachycardiaand frequent ventricular ectopic activity. No systemic hemodynamicchanges were noted after bFGF was administered. The areas at risk weresimilar in both groups (41±8 cm² versus 40±6 cm², vehicle versus bFGF).In the bFGF-treated group, infarct size expressed as a percentage of thearea at risk was 13.7±2.1%, which was significantly less than in dogsreceiving vehicle (28.4±3.4%; P=0.002; FIG. 3↓). At baseline, leftventricular ejection fractions were similar in both groups (bFGF versusvehicle, 42.6±1.9% versus 44.8±3.5%). After reperfusion (bFGF versusvehicle, 33.1±5.4% versus 40.3±3.2%) and again at 1 week afterinfarction (bFGF versus vehicle, 33.6±3.6% versus 38.8±3.5%), ejectionfractions showed no significant difference between groups (FIG. 4↓).

[0164] Microscopic examination of sections demonstrated concordancebetween triphenyltetrazolium chloride infarct delineation andhistological features of myocardial necrosis. Although bFGF treatmentwas associated with significant myocardial salvage, there was nodifference in the number of endothelial cells per high-power fieldwithin the infarcted region (bFGF versus vehicle, 241±16 versus 221±18cells/hpf; P=0.8) or in the number of endothelial cells in the borderzones (bFGF versus vehicle, 247±18 versus 245±15 cells/hpf; P=0.63).Because of the potential for spurious PCNA counts in areas of leukocyteinfiltration, PCNA counts were obtained from border zones only; thesecounts were similar in both groups (bFGF versus vehicle, 10.1±2.3 versus7.3±2.3 cells/hpf; P=0.4).

[0165] Measurement of Activated Clotting Time. Activated clotting timewas measured with the use of the Hemochron 801 timer (InternationalTechnidyne Corp). After 2 mL of whole blood was collected into aHemochron tube containing 12 mg of Johns-Manville diatomaceous earth,the time taken to complete coagulation at 37° C. was measured.

[0166] Delivery and Biological Activity of bFGF. To ensure delivery ofbFGF after passage through the manifold and angioplasty guidingcatheter, radiolabeled bFGF was passed through new and used systems. Tosimulate the conditions of an in vivo experiment, 20 μg of cold bFGF wasmixed with 25 μCi of radiolabeled bFGF in 20 mL of normal salinesolution. A second batch of 20 μg of cold bFGF was mixed with 25 ,μCi ofradiolabeled bFGF in 20 mL of normal saline solution containing 1 mg/mLof dog albumin (Sigma Chemical Co). The number of counts per minute fromboth solutions was quantified in a scintillation counter. Ten-milliliteraliquots of the radiolabeled solutions were then delivered through usedand new guiding catheters and manifolds and flushed with an additional10 mL of normal saline. The number of counts per minute in the solutionscollected after passage through the catheter system was measured. Thedifference in counts per minute between the incoming and outgoingsolution was used as an index of bFGF loss within the delivery system.Under the conditions described above, there was minimal loss of activityin the delivery system. The bFGF used in the experiments was compared ina mitogen assay with human recombinant bFGF from a commercial source(Boehringer Mannheim) that had proven activity in previous assays. Thepotency of both lots of bFGF was similar, as assessed by ³H-thymidineuptake after stimulation of cultured human fibroblasts (data not shown).

[0167] Determination of Infarct Size. After euthanasia and rapidexcision of the heart, the LAD and circumflex arteries were cannulatedindividually. Simultaneously, at a pressure of 100 mm Hg, the circumflexvessel was perfused with Evans blue dye and the LAD withtriphenyltetrazolium chloride for 10 minutes. Hearts were then fixed byperfusion with HistoChoice (Ameresco) for 4 hours, after which the leftventricle was cut into 1-cm-thick slices perpendicular to its long axis,and the slices were weighed. With this technique, areas of viable tissuein the LAD distribution are stained red, necrotic areas remain white,and the circumflex territory is stained blue. For each slice, the areaat risk, the area of infarction, and the circumflexterritory weredetermined by computer-assisted planimetry, as previously described.

[0168] Histology and Immunohistochemistry. Multiple tissue samples weretaken from areas of infarction and areas at risk of infarction forhistological examination to seek evidence of neovascularization. Giventhe assumptions that (1) neovascularization of ischemic regions wouldproceed from the circumflex and nonoccluded LAD distributions and (2)the tissue stimulus for neovascularization would be intense in tissueadjacent to the infarct zone, “border-zone” samples were taken from thearea at risk midway between the edges of the macroscopically infarctedmyocardium and the junction of the LAD and circumflex territories.Staining with hematoxylin and eosin was used to confirm the presence oftissue necrosis in the infarct zones. Immunohistochemical staining oftissue samples was performed with factor VIII-related antigen to detectendothelial cells and PCNA to detect proliferating cells.

[0169] After being embedded in paraffin, 5-μm sections were cut andcollected onto glass slides coated with 1% polychloroprene in xylene.After being dried for 60 minutes at 60° C., paraffin was removed inthree changes of xylene. The tissue was then rehydrated through gradedalcohols before being rinsed in PBS. Immunohistochemical staining wasperformed in a Jung Histostainer (Leica). A 0.6% hydrogen peroxidesolution in PBS was then applied for 5 minutes to remove any endogenousperoxidase. For the PCNA sections, a blocking solution of 1:10 (vol/vol)normal rabbit serum (Dako Corp) was added for 10 minutes beforeapplication of a 1-in-50 dilution of murine monoclonal antibodiesdirected against PCNA (PC10 Clone, Dako Corp). For the factorVIII-related antigen stain, a blocking solution of 1:10 (vol/vol) normalswine serum (Dako Corp) was added for 10 minutes before application of a1:300 dilution of rabbit polyclonal antibodies directed against factorVIII-related antigen (Dako Corp). The dilutions of the primaryantibodies were prepared with the use of 1% BSA in PBS and wereincubated with the tissue sample at 30° C. for 60 minutes. A 1:200dilution of biotinylated rabbit anti-mouse polyclonal antibody (DakoCorp) was then added for 30 minutes to the PCNA sections, and 1:200biotinylated swine anti-rabbit polyclonal antibody (Dako Corp) was addedto the factor VIII-stained sections for 30 minutes. These antibodieswere labeled with an Elite streptavidin-biotin-peroxidase complex(Vector Laboratories) applied for 30 minutes. The final stage involvedthe addition of 3,3′-diaminobenzidine (Vector Laboratories) as achromogen. Between steps, the sections were rinsed for 2 minutes in PBS.Slides were then rinsed in distilled water, dehydrated, cleared inxylene, and mounted in Permount (Fisher Scientific). In each stainingpreparation, sections treated with 1% BSA in PBS instead of with theprimary antibody were included as negative controls, and sections ofhuman tonsil were used as positive controls.

[0170] Cell Counts. Photographs of immunohistochemically stained tissuesections were taken without knowledge of treatment assignment. Afterlow-power examination, five to seven representative fields (0.5×0.34 mm)were photographed from each section at a magnification of 200×. Wheneverpossible, consecutive adjacent fields were photographed. In sectionsfrom the infarct zone, fields with relative preservation of tissuearchitecture were selected, obviating spurious increases in vesseldensity due to preservation of vascular structures in areas ofparenchymal loss and stromal collapse. Cells that stained positive forPCNA and factor VIII (regardless of the presence of a vascular lumen)were counted by two independent observers blinded to treatmentassignment (interobserver correlation coefficient, r=0.69; P<0.0001).Immunostaining for factor VIII and PCNA represented the techniquescurrently used as diagnostic tools for measurement of tumorangiogenesis.

[0171] Left Ventricular Ejection Fraction. Left ventricular ejectionfractions were determined from single-plane left ventriculogramsmeasured by a trained technician who was blinded to treatmentassignment. Ejection fractions were calculated by use of the length-areamethod with a computer analysis package (Angiographic VentricularDynamics 5.1, Siemens).

[0172] Acute Hemodynamic Studies In five additional dogs of either sex(weight, 19 to 22 kg), we compared the effects of intracoronary bFGF oncoronary hemodynamic parameters with those of temporary coronaryocclusion and intracoronary NTG. The studies were performed with the useof a standard open-chest model in which the LAD was isolated andinstrumented with a Doppler flow probe to measure blood flow (CrystalBiotech). A 2F catheter was advanced retrogradely via a small proximalbranch of the LAD into the left main vessel for administration of drugs.Blood flow responses after 10- and 20-second periods of LAD occlusionand after incremental doses of intracoronary NTG (1, 10, and 100 μg)were recorded to confirm the presence of coronary vascular reactivity.Incremental doses of intracoronary bFGF (1, 10, and 100 μg) were thengiven, and coronary flow responses were measured. bFGF (buffered asdescribed above) and NTG solutions were prepared in 1 mL of normalsaline just before administration and were given as boluses over 20seconds. Blood pressure, heart rate, and ECG were monitored continuouslythroughout the procedure. Coronary vascular resistance (CVR) wascalculated according to the formula

CVR (mm Hg·mL⁻¹)=mean aortic pressure (mm Hg)×1/coronary flow (mL/min)

[0173] The results of the occlusion-reperfusion study demonstrated areduction in infarct size without histochemical evidence of myocardialneovascularization. The acute hemodynamic study was performed to assessthe presence of a vasodilator action of bFGF as described in dogs andother species whereby flow to the infarct zone could possibly beauggmented by an increase in the conductance of preexisting collateralchannels, independently of neovascularization. In the five dogs studied,coronary blood flow and coronary vascular resistance were unchangedafter incremental pharmacological doses of intracoronary bFGF despitepronounced vasodilator responses to 10- and 20-second coronary occlusionand intracoronary NTG. In addition, three of the dogs were monitored for30 minutes after the final dose of bFGF (100 μg) to detect the presenceof a delayed vasodilator response as reported previously. No significanthemodynamic changes were observed in response to bFGF during theexperiment.

[0174] Institutional Approval and Sample Size. The protocol was approvedby the Cleveland Clinic Foundation Institutional Review Board and AnimalResearch Committee. Animals were handled in accordance with the NationalInstitutes of Health guidelines for the use of experimental animals. Inthe occlusion-reperfusion study, 22 dogs were randomized to receive bFGFor vehicle. Five dogs (2 treated with bFGF, 3 with vehicle) died ofarrhythmias before completion of the protocol. Three dogs (1 treatedwith bFGF, 2 with vehicle) were excluded because of persistent occlusionat the site of balloon occlusion. No dogs were excluded from the acutehemodynamic study.

[0175] Data Analysis. All data are expressed as mean±SEM. Differencesbetween groups were evaluated by use of two-tailed, unpaired t tests.The Pearson correlation coefficient was used to assess inter-observervariability for cell counts. Repeated measurements of left ventricularejection fraction were compared by use of two-way ANOVA. Differenceswere considered significant at a value of P<0.05.

[0176] This study has demonstrated that bFGF reduces the extent ofinfarction in the canine occlusion-reperfusion setting. Although thereis little doubt that the beneficial effects of bFGF on coronaryperfusion in chronic ischemia are mediated principally by its angiogenicactions, we have demonstrated that myocardial salvage occursindependently of neovascularization after administration of bFGF in thesetting of acute myocardial infarction. Further evaluation of coronaryvasomotor responses to bFGF in ischemic and nonischemic settings andinvestigation of the potential cytoprotective properties of bFGF inacute ischemia promise to provide fertile and clinically relevant areasfor future investigation.

Example 7 Intracoronary and Intravenous Administration of FGF-2

[0177] This study was designed to investigate the myocardial and tissuedeposition and retention of bFGF after IC and i.v. administration innormal and chronically ischemic animals.

[0178] Tissue distribution studies were carried out in 24 Yorkshire pigs(12 normal animals and 12 chronically ischemic animals). Yorkshire pigsof either sex weighing 15 to 18 kg were anesthetized with i.m. ketamine(10 mg/kg) and halothane inhalation anesthesia. By sterile technique, aright popliteal cut down was performed and a 4 French arterial catheterwas inserted for blood sampling and pressure monitoring. Leftthoracotomy was performed through the 4th intercostal space duringmechanical ventilation. The pericardium was opened and an ameroidconstrictor of 2.5 mm internal diameter (matched to the diameter of theartery) was placed around the proximal left circumflex coronary artery.The pericardium was closed using 6/0 Prolene and the chest was closed. Asingle dose of i.v. cefazolin (70 mg/kg) was given and i.m. narcoticanalgesics were administered as needed. Animals were then allowed torecover for 3 weeks (time sufficient for ameroid closure) beforeradiolabeled growth factor delivery. The treatment of animals was doneaccording to National Institutes of Health guidelines and the protocolwas approved by the Institutional Animal Care and Utilization Committeeof the Beth Israel Deaconess Medical Center.

[0179] A total of 24 animals was used for the study. Twelve animalsunderwent ameroid placement on the LCX, and 3 weeks later, afterconfirming LCX occlusion angiographically, received ¹²⁵-bFGF. IC¹²⁵I-bFGF was administered to six normal and six ischemic animals,whereas i.v. ¹²⁵I-bFGF was given to six normal and six ischemic animals.Tissue deposition was measured at 1 and 24 h in three animals of eachgroup. The use of these two time points was determined by the need tostudy more sustained myocardial deposition and retention of ¹²⁵I-bFGF.

[0180] Ischemic animals (three weeks after ameroid placement) and normalnoninstrumented animals were anesthetized with i.m. ketamine (10 mg/kg)and halothane inhalation anesthesia. By sterile technique, an i.v. linewas inserted into the ear vein and a right femoral cut down wasperformed to introduce an 8Fr arterial sheath. Coronary angiography wasthen performed in multiple views using a 7 French JR4 diagnosticcatheter (Cordis Laboratories, Inc., Miami, Fla.) to confirm LCXocclusion in ischemic animals and to assess the coronary anatomy.¹²⁵I-Bolton Hunter-labeled bFGF (¹²⁵I-bFGF; 25 μCi; New England Nuclear)with a specific activity of 110 μCi/μg (4050 kBq/μg) was combined with30 μg of cold bFGF and 3 mg of heparin (similar to the dose used inanimal studies and in the recent phase I IC and i.v. human study) andwas used for IC (six normal and six ischemic animals) and i.v. (sixnormal and six ischemic animals) delivery. For IC delivery, ¹²⁵I-bFGFwas infused in the left main coronary artery over 10 min. For i.v.delivery, ¹²⁵I-bFGF was infused through the ear vein i.v. line over 10min. Animals were then sacrificed 1 (n 12) and 24 h (n=12) after¹²⁵I-bFGF administration.

[0181] Extracardiac Deposition. Biodistribution of the i.v. and ICradiolabeled bFGF was determined at 1 and 24 h after administration andwas pooled for ischemic and nonischemic animals. There were nosignificant differences between ischemic and nonischemic animals at eachtime point and the data was therefore pooled. At 1 h, the liveraccounted for 37.6±17.1% of the total administered activity for IC and42.1±17.7% for i.v. delivery (p=0.6), with a reduction to 2.8±1.5% forIC and 1.5±0.9% for i.v. delivery by 24 h (p=0.09). Total specificactivity (1 h) in the kidneys was 2.3±1.3% for IC and 2.5±1.0% for i.v.delivery (p=0.8). By 24 h, total kidney specific activity decreased to0.1±0.05% for IC and 0.2±0.09 for i.v. delivery (p=0.1). Finally, for ICand i.v. delivery, total lung specific activity was 2.7±4.1 and 3.8±2.6%at 1 h (p=0.6) and 0.2±0.2 and 0.4±0.08% at 24 h (p=0.05), respectively.Specific activity for urine was 0.01±0.01% for IC and 0.005±0.01% fori.v. administration at 1 h and increased to 0.02±0.01% for IC and0.03±0.05% at 24 h for i.v. delivery, however, that increase was notstatistically significant.

[0182] Cardiac Deposition. Total specific activity (1 h) was 0.88±0.89%for IC and 0.26±0.08% for i.v. administration (p=0.12) and decreased to0.05±0.04% (p=0.05, compared with 1 h values) and 0.04±0.01% (p<0.001,compared with 1 h values) at 24 h, respectively. There were nodifferences between epicardial and endocardial deposition for both ICdelivery; the results were pooled for further analysis. For IC delivery,LAD territory activity per gram of tissue (1 h) was 0.01±0.007% and0.008±0.008% for normal and ischemic animals, and at 24h dropped to0.0005±0.0009% (20-fold reduction) in nonischemic animals and0.0008±0.0005% (10-fold reduction) in ischemic animals. For i.v.delivery, 1-h LAD territory activity per gram of tissue was 0.003±0.001%(3-fold reduction, p=0.2, compared with IC) and 0.002±0.0009% (4-foldreduction, p=0.3, compared with IC) for normal and ischemic animals, andat 24 h dropped to 0.0004±0.0001% (7.5-fold reduction) in nonischemicanimals and 0.0004±0.0004% (5-fold reduction) in ischemic animals,respectively. For 1-h LCX myocardial deposition, IC and i.v. deliveriesresulted in a specific activity per gram of tissue of 0.008±0.004% and0.003±0.001% (2.6-fold reduction, p=0.09) in normal animals and0.01±0.007% and 0.003±0.001% (3.3-fold reduction, p=0.2) in ischemicanimals, respectively. At 24 h, LCX deposition for IC and i.v. deliverydropped to 0.0006±0.0008% and 0.0005±0.0002% in normal animals and0.0006±0.0006% and 0.0004±0.0004% in ischemic animals, respectively. Forall groups, RCA myocardial distribution was similar to LAD and LCXdistribution for i.v. administration. However, for IC delivery, RCAmyocardial deposition was significantly lower than LAD or LCX myocardialdeposition, because the radiolabel was infused in the left main coronaryartery. Finally, for IC delivery, LCX/LAD territory activity was 79 and154% for nonischemic and ischemic animals at 1 h and 116% and 75% fornonischemic and ischemic animals at 24 h, respectively. Intravenousadministration resulted in an LCX/LAD activity of 97 and 100% fornonischemic and ischemic animals at 1 h and 1-23% and 98% fornonischemic and ischemic animals at 24 h, respectively.

[0183] Myocardial autoradiography confirmed myocardial deposition forboth IC and i.v. delivery with three times enhanced deposition for ICdelivery compared with i.v. delivery at 1 h with near equalization oftissue deposition at 24 h (measured using densitometric analysis). Inaddition, IC delivery resulted in increased deposition in LAD and LCXdeposition compared with RCA (noninfused territory) deposition, whereasi.v. delivery resulted in a more uniform distribution in the threemyocardial territories by qualitative analysis. Light levelautoradiography after 72-h exposure showed LAD endothelial depositionfor IC delivery after 1 h. Evaluation of other arteries for IC deliveryat 24 h and for all coronary arteries at all time points failed to show¹²⁵I-bFGF deposition even after 96 h of exposure.

[0184] Duplicate plasma, urine (spot samples), and tissue samples fromthe liver, lung, kidney, and quadriceps muscle were obtained. Tissueswere washed three times in saline to avoid contribution of radioactivityin blood. The heart, liver, lungs, and kidneys were weighed to determinetotal organ weight. Duplicate samples were also obtained from the rightventricle and from the proximal portion of the left anterior descendingcoronary arteries (LADs) and right coronary arteries (RCAs). A 1-cm midleft ventricular transverse slice was sectioned and cut into eightsegments; each segment was divided into epicardial, mid-myocardial, andendocardial portions. ¹²⁵I-bFGF activity was determined in a gammacounter (LKB Instruments, Inc., Gaithersburg, Md.). Background wassubtracted and the amount of ¹²⁵I-bFGF deposited within a specificsample was calculated as a percentage of the total activityadministered. Total solid organ deposition was calculated by multiplyingthe specific activity per gram of tissue by the weight of the organ.Trichloroacetic acid precipitation was performed to determine specificactivity, which averaged 86.3±24.4%. A 2-mm transverse left ventricularsection was obtained for organ level autoradiography and exposed in aphosphoimager for 24 h. In addition, tissue samples were obtained fromthe LAD and the subtended myocardium, formalin-fixed, paraffin-embedded,and 10 μm sections were mounted on a slide, coated by a photographicemulsion for 72 h, developed, and examined using light level microscopy.

[0185] Data are expressed as mean±S.D. Continuous variables werecompared by unpaired Student's t test, whereas categorical variableswere compared by X² analysis. All reported p values were two-tailed;p<0.05 was considered statistically significant.

[0186] Both IC and i.v. delivery strategies resulted in the majority ofradiolabel being deposited in the liver. Surprisingly, liver depositionwas similar for both techniques, indicating significant recirculationfor IC delivery. In addition, these results confirm the previousobservation that the liver is the major organ of elimination withcirculating bFGF binding to -2-macroglobulin, which in turn isinternalized by receptors on Kupffer. This result was duplicated forrenal and lung deposition. It is important to point out that bFGF wasinfused in the ear vein (above the diaphragm). However, this simulatesi.v. delivery in patients where the port of entry would probably be anupper extremity vein bypassing the liver first pass mechanism.Therefore, IC delivery does not result in less systemic deposition,probably due to high recirculation.

[0187] One-hour total and regional myocardial deposition was 3- to4-fold higher for IC compared with i.v. delivery, and deposition droppedby 5- to 20-fold at 24 h. IC delivery resulted in higher deposition inischemic myocardium, possibly related to the increased expression offibroblast growth factor receptors associated with myocardial ischemia.This was not seen in i.v. delivery, possibly related to the initialconcentrations delivered to the ischemic myocardium. Thus IC delivery,by providing higher initial concentrations in the coronary circulation,may result in higher deposition in ischemic areas. These comparisons,although consistent, did not reach statistical significance due to thesmall number of animals studied.

[0188] Of note, IC delivery resulted in enhanced bFGF depositioncompared with i.v. delivery only in myocardial territories subtended bythe infused artery. Therefore, for IC delivery to provide an advantageover i.v. delivery, infusion should be carried out in all coronaryarteries and bypass grafts if present. Whether infusing a larger dose ofbFGF would result in similar myocardial deposition to IC delivery (amore invasive approach) was not investigated. For IC delivery, bFGF wasidentified on the endothelial cells of the infused arteries, where itmight exert its effect. In addition, this study raises an importantquestion of whether more local or sustained delivery is necessary forbFGF effect, particularly with the relatively low cardiac deposition forboth delivery modalities.

Example 8 Administration of FGF, VEGF and Related Growth Factor Proteinsby Oral Inhalation

[0189] As demonstrated in the Example 7, above, intracoronary deliveryof FGF-2 resulted in enhanced FGF-2 deposition compared with i.v.delivery only in myocardial territories subtended by the infused artery.Also, both IC and i.v. delivery strategies resulted in the majority ofradiolabled protein being deposited in the liver. Given the presumptionthat the efficiency of delivery of therapeutic protein via pulmonaryadministration should be higher than for i.v. delivery, although perhapsnot as high as for more direct delivery routes (IC, interpericordial,intermyocardial), the dosage levels developed for IC delivery do notneed much adjustment for adaptation to delivery by oral inhalation. Inaddition, the ease of administration and the non-invasive nature of oralinhalation therapy permit the facile administration of repeated dosesshould initial dosing prove relatively ineffective. Thus, dosage levelsfor administration of FGF, VEGF and/or related growth factor proteinscan be initiated at levels substantially the same as those utilized forIC delivery. If ineffective, as measured by CPK-MB levels, then eitherrepeated doses, or higher dose loading can be utilized.

[0190] As discussed above, the preferred method of oral inhalationdelivery utilizes a dry powder inhalant formulation due to the relativehigh stability of proteins in the dried crystal form. However, as wouldbe recognized by one of skill in the appropriate art, it is possible toformulate the growth factor proteins in a solution for delivery throughoral inhalation of aerosol sprays using existing technology as developedfor other therapeutic proteins. See discussion of oral delivery ofinsulin, and patents referenced therein, above. In particular, incomparison to therapeutic species such as insulin, the deliveryparameters for FGF and similar proteins are much less stringent in thatthe severe, potentially fatal, consequences of administering overdosesof insulin are not of concern. Possible adverse consequences ofadministration are easily monitored on the clinical level andappropriate adjustments of dose level and dosing frequency can be made.

[0191] Accordingly, using technology and skills readily available to oneof ordinary skill in the pharmaceutical formulation arts, it is possibleto formulate a composition comprising FGF, VEGF and/or related proteinssuitable for delivery by oral inhalation, preferably in a dry powderform. Acceptable levels of dosing and a suitable dosing regimen aresummarized below: Protein Dose range (adult) Dosing Schedule FGF 10μg-20 mg 2 inhalations; repeat (acidic or basic) at five minutes; twicedaily; maintain for seven days VEGF 10 μg-10 mg 2 inhalations; repeat atfive minutes; twice daily; maintain for seven days

[0192] For acute conditions (MI, angina attack, unstable angina), twosets of double inhalations, five minutes apart should be attempted.Repeat, if necessary, only after assay of CPK-MB levels. Uponconfirmation of improvement in CPK-MB levels, remainder of seven-dayinitial dosing regimen can be resumed. Repeated administration should bediscontinued if bronchial spasms occur, or a decline in respiratoryfunction is detected.

[0193] Although the above is a complete description of the preferredembodiments of the invention, various alternatives, modifications, andequivalents may be used, as recognized by one of ordinary skill in theappropriate art, based upon the teachings disclosed herein. Therefore,the above description should not be taken as limiting the scope of theinvention that is defined by the claims below.

What is claimed is:
 1. A method for the systematic multi-tieredtreatment of heart disease by delivery of therapeutic growth factorproteins comprising the steps of: a.) selecting a patient displayingsymptoms of heart disease; b.) administering at least one dose of aneffective amount of a first therapeutic growth factor proteinformulation by oral inhalation; c.) monitoring levels of CPK-MB in thepatient; d.) determining whether administration of the growth factorprotein formulation was effective in treating the symptoms of heartdisease in the patient; e.) administering one or more additional dosesof a second growth factor protein formulation by a method of deliverymore invasive than delivery by oral inhalation; and f.) repeating stepsc.) through e.) until there is a clinical indication of amelioration ofthe symptoms of heart disease in the patient, or until there is acontraindication to continued treatment.
 2. The method of claim 1,wherein the protein formulation comprises a growth factor proteinselected from the group consisting of FGF-1, FGF-2, VEGF, and mixturesthereof.
 3. The method of claim 1 wherein the symptoms of heart diseaseare acute.
 4. The method of claim 3 wherein the acute symptoms of heartdisease are brought on by a condition selected from the group consistingof myocardial infarct, unstable angina, an acute anginal attack, andreperfusion injury.
 11. The method of claim 10, wherein the proteinformulation comprises a growth factor protein selected from the groupconsisting of FGF-1, FGF-2, VEGF, and mixtures thereof.
 12. The methodof claim 10 wherein the heart disease is characterized by acutesymptoms.
 13. The method of claim 12 wherein the acute symptoms of heartdisease are brought on by a condition selected from the group consistingof myocardial infarct, unstable angina, an acute anginal attack, andreperfusion injury.
 14. The method of claim 13, wherein the reperfusioninjury is induced by a procedure selected from the group consisting ofthrombolytic therapy, bypass surgery and angioplasty.
 15. The method ofclaim 10 wherein the heart disease is characterized by symptoms that arechronic.